Plant tolerance to stress through the control of chloroplast stability

ABSTRACT

The chloroplast vesiculation (CV) gene encodes a protein associated with stress-induced chloroplast degradation. Inhibiting expression or activity of the protein inhibits or delays stress-induced chloroplast degradation and confers tolerance to a variety of stress conditions. Alternatively, enhancing CV expression or activity promotes chloroplast degradation and enhances nutrient assimilation in desired sink tissues, such as young leaves, fruit, or seeds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C.§119(e) to U.S.Application No. 61/897,006, filed Oct. 29, 2013, the contents of whichare incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to plants having increased tolerance to stress orhaving enhanced nutrient assimilation in sink tissues, and methods ofpreparing the plants.

BACKGROUND OF THE INVENTION

Environmental stress such as high salinity, extreme temperatures, anddrought are responsible for high yield loses of major crops worldwide(Mittler and Blumwald 2010, Annual Review Of Plant Biology 61: 443-462).Plants use an escape strategy to cope with stress, which ischaracterized by early flowering and leaf senescence (Levitt 1972, AnnuRev Plant Biol 58: 115-136; Ludlow 1989, Strategies in response to waterstress. SPB Academic Press, The Netherlands; Mittler and Blumwald 2010,Annual Review Of Plant Biology 61: 443-462). During leaf senescence, theearliest event is the degradation of the chloroplasts that possess up to70% of total leaf proteins (Lim et al. 2007; Ishida et al. 2008, PlantPhysiol 148: 142-155). The mobile nitrogen resulting from chloroplastdisassembly is recycled and supplied to the sink organs, flowers andseeds (Liu et al. 2008 J Plant Biol 51: 11-19). However, thestress-induced chloroplast degradation and premature senescence canaffect plant photosynthetic capacity and eventually compromise the cropyield.

Although the inhibition of photosynthetic activity and the degradationof the photosynthetic apparatus are a primary target of abiotic stresses(Rivero et al. 2007, Proceedings of the National Academy of Sciences ofthe United States of America 104: 19631-19636), the mechanisms ofstress-induced chloroplast degradation remain largely unknown. As anindispensable step of chloroplast degradation, the chlorophyll breakdownhas been investigated in detail in Arabidopsis (Hortensteiner 2009,Trends Plant Sci 14: 155-162). Five chlorophyll catabolic enzymes thatcatalyze green chlorophyll to colorless nonfluorescent chlorophyllcatabolites, which are finally disposed in the vacuole, have beenidentified (Hortensteiner 2006 Annual Review of Plant Biology 57: 55-77;Hortensteiner 2009 Trends Plant Sci 14: 155-162); Sakuraba et al. 2012,Plant Cell 24: 507-518). Recently, SGR a gene encoding a nonenzymeprotein SGR (stay-green) has been shown to be a key factor inchlorophyll degradation (Jiang et al. 2007; Park et al. 2007, Plant Cell19: 1649-1664; Ren et al. 2007, Plant Physiology 144: 1429-1441). InArabidopsis, the SGR protein (AtNYE1) was able to destabilize thelight-harvesting complex II (LHCII) and recruited the five chlorophyllcatabolic enzymes to the thylakoids of senescing chloroplast forchlorophyll degradation. After the chlorophyll degradation, thechlorophyll-binding proteins were more susceptible to digestion bychloroplast proteases (Park et al. 2007; Ren et al. 2007, PlantPhysiology 144: 1429-1441; Hortensteiner 2009; Sakuraba et al. 2012,Plant Cell 24: 507-518).

Two pathways have been demonstrated for the degradation of chloroplaststromal proteins: autophagy (Ishida and Yoshimoto 2008 Autophagy 4:961-962; Ishida et al. 2008, Plant Physiol 148: 142-155; Wada et al.2009, Plant Physiol 149: 885-893; Izumi et al. 2010, Plant Physiol 154:1196-1209) and senescence-associated vacuoles (SAV) (Otegui et al. 2005,Plant Journal 41: 831-844; Martinez et al. 2008, Plant Journal 56:196-206; Carrion et al. 2013). Autophagy is a well-known system for thebulk degradation of intracellular proteins and organelles (Ohsumi 2001,Nature Reviews Molecular Cell Biology 2: 211-216; Bassham 2009, BiochimBiophys Acta 1793: 1397-1403). Plant autophagy has been shown tofunction in senescence, defense against pathogens and response toabiotic stress (Bassham, 2009 Biochim Biophys Acta 1793: 1397-1403;Reumann et al. 2010, Protoplasma 247: 233-256; Liu and Bassham 2012,Annu Rev Plant Biol 63: 215-237). The chloroplast Rubisco protein andstroma-targeted fluorescent proteins were shown to move to the vacuolevia autophagic bodies named Rubisco-containing bodies (RCBs).Dark-induced chloroplast degradation and RCBs formation were impaired inautophagy-defective mutants (Ishida and Yoshimoto 2008, Autophagy 4:961-962; Ishida et al. 2008, Plant Physiol 148: 142-155; Wada et al.2009, Plant Physiol 149: 885-893). Even whole chloroplasts have beenshown to be transported to the vacuole through the autophagy-dependentprocess in individually darkened leaves (Ishida and Wada 2009 Autophagy5: 736-737; Wada et al. 2009, Plant Physiol 149: 885-893).Interestingly, RCBs-mediated chloroplast degradation was highlyactivated by the shortage of carbon source rather than nitrogen source(Izumi et al. 2010, Plant Physiol 154: 1196-1209; Izumi and Ishida 2011,Plant Signal Behav 6: 685-687). This observation might be partiallyexplained by studies showing that autophagy also participates inchloroplast starch degradation by engulfing small starch granule-likestructures from chloroplast and transporting them to the vacuole forsubsequent breakdown (Wang et al. 2013, Plant Cell 25: 1383-1399).

In spite of the increasing information regarding processes associatedwith the degradation of chloroplast stroma proteins, the pathway(s) bywhich thylakoid membrane proteins are released from the chloroplast andtransported to the vacuole for degradation remain poorly understood.Thus, the identification and characterization of genes associated withchloroplast destabilization would be useful to develop new plantvarieties with altered source/sink interactions and thus provideenhanced nutrient assimilation in desired tissues in the plant. Inaddition, identification of such genes would also be useful to developnew plant varieties in which chloroplast degradation is inhibited ordelayed, thus conferring tolerance to stress conditions on the plants.This invention addresses these and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of preparing a transgenic planthaving enhanced stress tolerance. The methods comprise introducing intoa population of plants an expression cassette that inhibits expressionof a chloroplast vesiculation (CV) gene. In a typical embodiment, themethod further includes the step of selecting a plant having enhancedstress tolerance compared to a control plant that does not comprise theexpression cassette. The step of introducing the expression cassette canbe carried out by any known method such as, for example, usingAgrobacterium.

In a typical embodiment, the expression cassette comprises a nucleicacid sequence encoding a microRNA or an siRNA specific to the CV gene.The target CV gene may encode a CV protein comprising the consensussequence RxCxxWxxN (SEQ ID NO: 45) and/or the consensus sequenceExxxPENLPRxxxxxR (SEQ ID NO: 46), where “x” can be any amino acid.Alternatively, the CV gene can encode a polypeptide comprising an aminoacid sequence at least 90% identical to any one of SEQ ID NOs: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,or 44. The target CV gene may comprise a coding sequence at least 90%identical to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43.

The expression cassette may comprise a constitutive promoter or aninducible promoter. The inducible promoter may be one that is induced inresponse to stress conditions.

In some embodiments, the transgenic plant has enhanced tolerance to anabiotic stress, such as high salt conditions. In these embodiments, thestep of selecting includes selecting plants having enhanced salttolerance.

In some embodiments, the abiotic stress is drought conditions. In theseembodiments, the step of selecting includes selecting plants havingenhanced drought tolerance.

The invention also provides plants prepared by the above methods of theinvention.

The invention further provides isolated nucleic acid moleculescomprising a plant promoter operably linked to a nucleic acid sequenceencoding a microRNA or an siRNA specific to a target CV gene. The targetCV gene typically encodes a CV protein comprising a consensus sequenceas shown in SEQ ID NO: 45 and/or SEQ ID NO: 46 or encodes a polypeptidecomprising an amino acid sequence at least 90% identical to any one ofSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, or 44. The target CV gene may comprise a codingsequence at least 90% identical to any one of SEQ ID NOs: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43.The plant promoter may be a constitutive promoter or an induciblepromoter.

In other embodiments, the invention provides methods of preparing atransgenic plant having enhanced stress tolerance. These methodscomprise (a) introducing mutations in CV genes in a population ofplants; and optionally (b) selecting a plant having enhanced stresstolerance compared to a control plant that does not comprise themutation.

The mutations may be introduced into the plant using chemicalmutagenesis. The desired plants can be identified using TargetingInduced Local Lesions in Genomes (TILLING). The invention also providesplants prepared by these methods.

The invention further provides transgenic plants comprising anexpression cassette comprising a plant promoter operably linked to anucleic acid sequence encoding a microRNA or an siRNA specific to atarget CV gene. The target CV gene may encode a CV protein comprising aconsensus sequence as shown in SEQ ID NO: 45 and/or SEQ ID NO: 46 orencode a polypeptide comprising an amino acid sequence at least 90%identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44. The target CV genemay comprise a coding sequence at least 90% identical to any one of SEQID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, or 43.

The invention also provides methods of preparing a transgenic planthaving enhanced nutrient assimilation. The methods comprise introducinginto a population of plants an expression cassette comprising aninducible plant promoter operably linked to a CV polynucleotide sequenceencoding a polypeptide at least 90% identical to any one of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, or 44. In the typical embodiment, the methods further compriseselecting a plant having enhanced nutrient assimilation compared to acontrol plant that does not comprise the expression cassette. The CVpolynucleotide may comprise a coding sequence at least 90% identical toany one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, or 43. The step of introducing theexpression cassette may be carried using Agrobacterium. The step ofselecting may be carried out by selecting plants with increased fruityield compared to control plants. The invention also provides transgenicplants prepared by these methods.

The invention also provides an expression cassette comprising aninducible plant promoter operably linked to a CV polynucleotide sequenceencoding a CV protein comprising a consensus sequence as shown in SEQ IDNO: 45 and/or SEQ ID NO: 46 or encoding a polypeptide comprising anamino acid sequence at least 90% identical to any one of SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, or 44. The CV polynucleotide may comprise a coding sequence at least90% identical to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43. The inventionfurther provides plants comprising the expression cassettes.

DEFINITIONS

As used herein, a “stress” (either abiotic or biotic) refers to theexposure of a plant to an agent (living or non-living) or condition thathas an adverse effect on metabolism, development, propagation, and/orsurvival of the plant (collectively “growth”).

As used herein, the terms “abiotic stress” or “abiotic stress condition”refer to exposure of a plant to a non-living physical or chemical agentor condition that has an adverse effect on the growth of the plant. Sucha stress can be imposed on a plant due, for example, to an environmentalfactor such as excessive or insufficient water (e.g., flooding, drought,dehydration), anaerobic conditions (e.g., a low level of oxygen),abnormal osmotic conditions, salinity or temperature (e.g., hot/heat,cold, freezing, frost), a deficiency of nutrients or exposure topollutants, or by a hormone, second messenger or other molecule.Anaerobic stress, for example, is due to a reduction in oxygen levels(hypoxia or anoxia) sufficient to produce a stress response. A floodingstress can be due to prolonged or transient immersion of a plant, plantpart, tissue or isolated cell in a liquid medium such as occurs duringmonsoon, wet season, flash flooding, excessive irrigation of plants, orthe like. A cold stress or heat stress can occur due to a decrease orincrease, respectively, in the temperature from the optimum range ofgrowth temperatures for a particular plant species. Such optimum growthtemperature ranges are readily determined or known to those skilled inthe art. Dehydration stress can be induced by the loss of water, reducedturgor, or reduced water content of a cell, tissue, organ or wholeplant. Drought stress can be induced by or associated with thedeprivation of water or reduced supply of water to a cell, tissue, organor organism. Saline stress (salt-stress) can be associated with orinduced by a perturbation in the osmotic potential of the intracellularor extracellular environment of a cell. Osmotic stress also can beassociated with or induced by a change, for example, in theconcentration of molecules in the intracellular or extracellularenvironment of a plant cell, particularly where the molecules cannot bepartitioned across the plant cell membrane.

As used herein, the term “drought stress” refers to conditions in whichevapotranspiration demand for water exceeds the supply of water. Droughttolerant plants of the invention will show better growth and/or recoveryfrom the stress, as compared to drought sensitive (e.g., control)plants. Typically, the drought stress will be at least 5 days and can beas long as 18 to 20 days with little or no added water.

The term “water-use efficiency” refers to the productivity of a plantper unit of water applied. For example, a plant may grow withsubstantially no yield penalty under extended periods with less thannormal (typically about half) amounts of water.

As used herein, the term “salt stress” refers to conditions in whichsalinity has an adverse effect on growth of a plant. While for eachspecies, the threshold at which soil and/or water salinity differs, asalt-tolerant plant will have a higher salinity threshold before growthor other measures of productivity decline, as compared to a control orreference plant.

As used herein, the term “biotic stress” refers to stress that occurs asa result of damage caused to plants by other living organisms, such asbacteria, viruses, fungi, parasites, insects, birds, mammals or otherplants.

A “chloroplast vesiculation (CV) gene” or a “CV polynucleotide” is agene or nucleic acid sequence (DNA or RNA) comprising at least a portionof a coding region which encodes a CV protein of the invention. A CVpolynucleotide may also be an RNA molecule (e.g., short intefering RNA,or micoRNA) transcribed from a CV DNA. The CV polynucleotide maycomprise a coding sequence at least 90% identical to any one of SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, or 43.

A “chloroplast vesiculation (CV) polypeptide” or “CV protein” is apolypeptide or protein which is at least substantially identical to anyone of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, or 44 and which controls chloroplaststability in plant cells. CV polypeptide or protein can also beidentified by the presence of the consensus sequence as shown here. Insome embodiments, the CV polypeptide or protein may comprise either orboth of the following consensus sequences: RxCxxWxxN (SEQ ID NO: 45) orExxxPENLPRxxxxxR (SEQ ID NO: 46), where “x” can be any amino acid. A CVpolypeptide of the invention typically comprises about 50 to about 195amino acids, often between about 100 and about 150 amino acids.

The phrase “nucleic acid” or “polynucleotide sequence” refers to asingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases read from the 5′ to the 3′ end. Nucleic acids mayalso include modified nucleotides that permit correct read through by apolymerase and do not alter the expression of a polypeptide encoded bythat nucleic acid.

The term “promoter” refers to a region or sequence determinants locatedupstream or downstream from the start of transcription and which areinvolved in recognition and binding of RNA polymerase and other proteinsto initiate transcription. A “plant promoter” is a promoter capable ofinitiating transcription in plant cells. Such promoters need not be ofplant origin, for example, promoters derived from plant viruses, such asthe CaMV35S promoter, can be used in the present invention.

The term “constitutive” or “constitutively” denotes temporal and spatialexpression of the CV polypeptides or nucleic acids of the presentinvention in plants in the methods according to various exemplaryembodiments of the invention. The term “constitutive” or“constitutively” means the expression of the polypeptides or nucleicacids of the present invention in the tissues of the plant throughoutthe life of the plant and in particular during its entire vegetativecycle. In some embodiments, the polypeptides or nucleic acids areexpressed constitutively in all plant tissues. In some embodiments, thepolypeptides or nucleic acids are expressed constitutively in the roots,the leaves, the stems, the flowers, and/or the fruits. In otherembodiments of the invention, the polypeptides or nucleic acids areexpressed constitutively in the roots, the leaves, and/or the stems.

The term “inducible” or “inducibly” means the CV polypeptides or nucleicacids of the present invention are not expressed, or are expressed atvery low levels, in the absence of an inducing agent. The expression ofthe polypeptides of the present invention is greatly induced in responseto an inducing agent.

The term “inducing agent” is used to refer to a chemical, biological orphysical agent or environmental condition that effects transcriptionfrom an inducible regulatory element. In response to exposure to aninducing agent, transcription from the inducible regulatory elementgenerally is initiated de novo or is increased above a basal orconstitutive level of expression. Such induction can be identified usingthe methods disclosed herein, including detecting an increased level ofRNA transcribed from a nucleotide sequence operatively linked to theregulatory element, increased expression of a polypeptide encoded by thenucleotide sequence, or a phenotype conferred by expression of theencoded polypeptide.

The term “plant” includes whole plants, shoot vegetativeorgans/structures (e.g. leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g. bracts, sepals, petals, stamens, carpels,anthers and ovules), seeds (including embryo, endosperm, and seed coat)and fruit (the mature ovary), plant tissue (e.g. vascular tissue, groundtissue, and the like) and cells (e.g. guard cells, egg cells, trichomesand the like), and progeny of same. The class of plants that can be usedin the method of the invention is generally as broad as the class ofhigher and lower plants amenable to transformation techniques, includingangiosperms (monocotyledonous and dicotyledonous plants), gymnosperms,ferns, bryophytes, and multicellular algae. It includes plants of avariety of ploidy levels, including aneuploid, polyploid, diploid,haploid and hemizygous.

The phrase “host cell” refers to a cell from any organism. Preferredhost cells are derived from plants, bacteria, yeast, fungi, insects orother animals. Methods for introducing polynucleotide sequences intovarious types of host cells are well known in the art.

A polynucleotide sequence is “heterologous to” a second polynucleotidesequence if it originates from a foreign species, or, if from the samespecies, is modified by human action from its original form. Forexample, a promoter operably linked to a heterologous coding sequencerefers to a coding sequence from a species different from that fromwhich the promoter was derived, or, if from the same species, a codingsequence which is different from any naturally occurring allelicvariants.

A polynucleotide “exogenous to” an individual plant is a polynucleotidewhich is introduced into the plant, or a predecessor generation of theplant, by any means other than by a sexual cross. Examples of means bywhich this can be accomplished are described below, and includeAgrobacterium-mediated transformation, biolistic methods,electroporation, in planta techniques, and the like.

The term “expression cassette” refers to any recombinant expressionsystem for the purpose of expressing a CV nucleic acid sequence of theinvention in vitro or in vivo, constitutively or inducibly, in any cell,including, in addition to plant cells, prokaryotic, yeast, fungal,insect or mammalian cells. The expression cassettes of the inventiontypically comprise a plant promoter operably linked to a CVpolynucleotide. The expression cassettes can be used to transcribe RNAmolecules that inhibit endogenous CV expression or to encode CVpolypeptides that enhance CV activity in the host cell.

In the case where the inserted CV polynucleotide sequence is transcribedand translated to produce a functional CV polypeptide, one of skill willrecognize that because of codon degeneracy, a number of polynucleotidesequences will encode the same polypeptide. These variants arespecifically covered by the term “CV polynucleotide”. In addition, theterm specifically includes sequences (e.g., full length sequences)substantially identical (determined as described below) with a CV genesequence encoding a CV polypeptide of the invention.

In the case of polynucleotides used to express CV RNA molecules thatinhibit expression of an endogenous CV gene, the introduced sequenceneed not be perfectly identical to a sequence of the target endogenousgene. The introduced polynucleotide sequence will typically be at leastsubstantially identical (as determined below) to the target endogenoussequence.

Two nucleic acid sequences or polypeptides are said to be “identical” ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The term “complementary to” is used herein to mean thatthe sequence is complementary to all or a portion of a referencepolynucleotide sequence.

Optimal alignment of sequences for comparison may be conducted by thelocal homology algorithm of Smith and Waterman, Add. APL. Math. 2:482(1981), by the homology alignment algorithm of Needleman and Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearsonand Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, BLAST,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

“Percentage of sequence identity” is determined by comparing twooptimally aligned polynucleotide or polypeptide sequences over acomparison window, wherein the portion of the sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity.

The term “substantial identity” of polynucleotide or polypeptidesequences means that a polynucleotide or polypeptide comprises asequence that has at least 85% sequence identity to a referencepolynucleotide or polypeptide sequence. In the case of CV polypeptidesof the invention, the reference polypeptide sequence can be any one ofSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, or 44. In the case of CV polynucleotides of theinvention, the reference polynucleotide sequence can be any one of SEQID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, or 43. More preferred embodiments include CVpolypeptides or polynucleotides at least 90%, 95%, or 99% compared to areference sequence (e.g., any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44, or anyone of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, or 43) using the programs described herein;preferably BLAST using standard parameters, as described below.Accordingly, polynucleotide sequences encoding a CV polypeptide used inthe methods of the present invention include nucleic acid sequences thathave substantial identity to the sequences disclosed here. One of skillwill recognize that these values can be appropriately adjusted todetermine corresponding identity of proteins encoded by two nucleotidesequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning and the like. CV polypeptides thatare “substantially similar” share sequences as noted above except thatresidue positions which are not identical may differ by conservativeamino acid changes. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.

Another indication that two CV nucleotide sequences are substantiallyidentical is if the two molecules hybridize to each other, or areference CV polynucleotide (e.g, any one of SEQ ID NOs: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43)under stringent conditions. Stringent conditions are sequence dependentand will be different in different circumstances. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength and pH.The Tm is the temperature (under defined ionic strength and pH) at which50% of the target sequence hybridizes to a perfectly matched probe.Typically, stringent conditions will be those in which the saltconcentration is about 0.02 molar at pH 7 and the temperature is atleast about 60° C. or 65° C.

For the purposes of this disclosure, stringent conditions forhybridizations are those which include at least one wash in 0.2×SSC at63° C. for 20 minutes, or equivalent conditions. Moderately stringentconditions include at least one wash (usually 2) in 0.2×SSC at atemperature of at least about 50° C., usually about 55° C., for 20minutes, or equivalent conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that artificial miRNA silencing of AtCV delayed thechloroplast degradation induced by salt stress. (A) Quantitative RT-PCRanalysis of AtCV expression in all leaves from 30-day-old plants ofCol-0 and three independent artificial-microRNA-silenced lines of AtCV(amiR-1, amiR-2 and amiR-3). (B) leaf chlorophyll content of Col-0 andthree independent AtCV-silenced lines (amiR-1, -2, -3) during saltstress treatment (50 mM NaCl for 3 days, 100 mM NaCl for 3 days, 150 mMNaCl for 10 days). Chlorophyll was extracted from leaf tissues. Mean±SDvalues were obtained from three independent experiments. Asteriskindicates P<0.001.

FIG. 2 shows that silencing AtCV increased drought tolerance intrangsenic plants. Plants of Wild type Col-0 and AtCV-silencedtransgenic lines (amiR-1, -2 and -3) were subjected to water stress for14 days and rewatering for 4 days. (A) Survival rate was determined 4days after rewatering. 15 plants for each line were evaluated andMean±SD were obtained from three independent experiments. Asterisk meansP<0.001. (B) AtCV expression in leaves from Col-0 and amiR-1 plantsduring drought treatment were determined by quantitative PCR.

FIG. 3 shows the results of drought stress experiments carried out usingtransgenic rice plants expressing amiRNAs of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based at least in part on the discovery that modulationof CV expression and activity in plants can be used to confer desirabletraits on plants. As shown below, CV proteins are associated withstress-induced chloroplast degradation. Thus, inhibiting expression oractivity of the protein will inhibit or delay stress-induced chloroplastdegradation and will confer tolerance to a variety of stress conditions.Alternatively, enhancing CV expression or activity will promotechloroplast degradation and will enhance nutrient assimilation indesired sink tissues, such as young leaves, fruit, or seeds.

Thus, in some embodiments, the present invention provides plants (whichcan be transgenic or non-transgenic) in which expression of theendogenous CV gene is inhibited. Such plants are more tolerant to astress condition (abiotic or biotic) than a corresponding control orreference plant. As used herein, the term “tolerant” when used inreference to a stress condition of a plant, means that the particularplant, when exposed to a stress condition, shows less of an effect, orno effect, in response to the condition as compared to a correspondingcontrol or reference plant (i.e., a naturally occurring wild-type plantor a plant not containing a construct of the present invention). As aconsequence, a plant of the present invention shows improved agronomicperformance (such as increased biomass, higher yields, and/or more seedproduction) as a result of enhanced abiotic or biotic stress toleranceand grows better under more widely varying conditions. Preferably, theplant is capable of substantially normal growth under environmentalconditions where the corresponding control or reference plant showsreduced growth, yield, metabolism or viability, or increased male orfemale sterility.

A plant's response to abiotic stress includes the production of excessreactive oxygen species (ROS), including singlet oxygen, superoxide,hydrogen peroxide and hydroxyls radicals, which act as signalingmolecules and play a role in the initiation of defense mechanisms. ROSare involved in wide variety of environmental stresses in plants.Excessive temperature extremes, water stress, ion imbalances due tosalinity, air pollution, and mechanical damage lead to chemical signalspropagated through ROS. Adaptation to the stress involves quenching ofROS signal through one or more anti-oxidant enzymes or compounds, suchas superoxide dismutase (SOD), glutathione, ascorbate, carotenoids, andothers. When the plant's quenching systems are exceeded by theenvironmental stress, extensive and rapid degeneration reactions canoccur through ROS, such as protein denaturation and lipid peroxidation.Thus, one of skill will recognize that improved tolerance to oneparticular type of abiotic stress, such as drought or salt, can beindicative of a similarly improved tolerance to other types of abioticstress.

In other embodiments, the invention provides plants in which CVexpression and/or activity is enhanced and which have enhanced nutrientassimilation in desired sink tissues in the plant. Within a plant, a“source” may be defined as a tissue or organ (usually a photosynthetictissue or organ, such as a leaf) which exports sugars and othernutrients to a “sink” tissue (usually a storage root, tuber, fruit seed,or young organ). As discussed above, during senescence, source tissuesare the site of the degradation of chloroplast proteins through theactivation of various chloroplast proteases. The protease products arethen mobilized into vesicles via extensive vesicular trafficking toyoung tissues (sinks), where nitrogen and other nutrients are used forbiosynthetic processes. Thus, plants having enhanced CV expressionand/or activity provide more nutrients to sink tissues such as storageroots, tubers, young organs, fruits and seeds.

For example in a typical embodiment, an expression cassette comprising aCV polynucleotide operably linked to a chemically induced promoter isintroduced into a desired plant (e.g., a tomato plant). At the time offruit set, the plant is treated with a chemical that induces expressionof the CV polynucleotide to induce chloroplast degradation in desiredtissues in the plant (e.g., leaves adjacent to the fruit). Nitrogen andother nutrients resulting from the degradation of chloroplasts in theleaves are then assimilated by the fruit, thereby enhancing thenutritional content of the fruit.

As demonstrated below, CV proteins are highly conserved in the plantkingdom. Thus, one of skill will recognize the CV genes and proteinsfrom a wide variety of plants can be used in the present invention. Theproteins can be identified by the presence of the consensus sequence asshown here. In particular, a CV protein can be identified by thepresence of either or both of the following consensus sequences:RxCxxWxxN (SEQ ID NO: 45) or ExxxPENLPRxxxxxR (SEQ ID NO: 46), where “x”can be any amino acid.

The invention has use over a broad range of plants, including speciesfrom the genera Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus,Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza,Panieum, Pannesetum, Persea, Pisum, Populus, Pyrus, Prunus, Raphanus,Secale, Senecio, Sinapis, Solanum, Sorghum, Theobroma, Trigonella,Triticum, Vitis, Vigna, and Zea.

CV Polynucleotides and Expression Cassettes

The present invention provides isolated nucleic acid moleculescomprising a CV polynucleotide of the present invention. Thepolynucleotide can be, for example, a DNA molecule that encodes a CVpolypeptide or an RNA molecule that inhibits endogenous CV expression ina cell.

The isolated nucleic acids of the present invention can be made usingstandard recombinant methods, synthetic techniques, combinationsthereof, or any method known to those of skill in the art.

The isolated nucleic acid compositions of this invention can be obtainedfrom plant biological sources (e.g., tissues from the plant) or can beprepared by direct chemical synthesis using any number of methodologiesfamiliar to those of skill in the art. In some embodiments,oligonucleotide probes that selectively hybridize under stringentconditions to the polynucleotides of the present invention are used toidentify the desired CV sequence in a cDNA or genomic DNA library.Isolation of RNA and construction of cDNA and genomic libraries is wellknown to those of ordinary skill in the art.

The nucleic acids of interest can also be amplified from nucleic acidsamples using amplification techniques. For instance, polymerase chainreaction (PCR) technology can be used to amplify the sequences ofpolynucleotides of the present invention and related genes directly fromgenomic DNA or cDNA libraries. PCR and other in vitro amplificationmethods may also be useful, for example, to clone nucleic acid sequencesthat code for proteins to be expressed, to make nucleic acids to use asprobes for detecting the presence of the desired mRNA in samples, fornucleic acid sequencing, or for other purposes.

The present invention also provides recombinant expression cassettescomprising a CV polynucleotide. Such plant expression cassettestypically contain the CV polynucleotide operably linked to a promoter(e.g., one conferring inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal. For example, a cDNA or a genomic sequenceencoding a full length a CV polypeptide, can be used to construct arecombinant expression cassette, which can be used to produce a CVprotein in a desired host cell. Alternatively, the expression cassettemay encode an RNA molecule that inhibits expression of an endogenous CVgene in the host cell. A recombinant expression cassette will typicallycomprise a polynucleotide of the present invention operably linked totranscriptional initiation regulatory sequences, which will direct thetranscription of the polynucleotide in the intended host cell, such astissues of a transformed plant.

A number of promoters can be used in the practice of the invention. Aplant promoter fragment can be employed which will direct expression ofthe CV polynucleotide in all tissues of a regenerated plant. Suchpromoters are referred to herein as “constitutive” promoters and areactive under most environmental conditions and state of development orcell differentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region.

Alternatively, the plant promoter can direct expression of thepolynucleotide under environmental control. Such promoters are referredto here as “inducible” promoters. Environmental conditions that mayaffect transcription by inducible promoters include biotic stress,abiotic stress, saline stress, drought stress, pathogen attack,anaerobic conditions, cold stress, heat stress, hypoxia stress, or thepresence of light.

In addition, chemically inducible promoters can be used. Examplesinclude those that are induced by benzyl sulfonamide, tetracycline,abscisic acid, dexamethasone, ethanol or cyclohexenol.

Examples of promoters under developmental control include promoters thatinitiate transcription only, or preferentially, in certain tissues suchas leaves, roots, fruit, seeds, or flowers. These promoters aresometimes called tissue-preferred promoters. The operation of a promotermay also vary depending on its location in the genome. Thus, adevelopmentally regulated promoter may become fully or partiallyconstitutive in certain locations. A developmentally regulated promotercan also be modified, if necessary, for weak expression.

As noted above, the invention provides a method of suppressing CVexpression or activity in a plant using expression cassettes thattranscribe CV RNA molecules that inhibit endogenous CV expression oractivity in a plant cell. Suppressing or silencing gene function refersgenerally to the suppression of levels of CV mRNA or CV proteinexpressed by the endogenous CV gene and/or the level of the CV proteinfunctionality in a cell. The terms do not specify mechanism and couldinclude RNAi (e.g., short interfering RNA (siRNA) and micro RNA(miRNA)), anti-sense, cosuppression, viral-suppression, hairpinsuppression, stem-loop suppression, CRSIPR, and the like.

A number of methods can be used to suppress or silence gene expressionin a plant. The ability to suppress gene function in a variety oforganisms, including plants, using double stranded RNA is well known.Expression cassettes encoding RNAi typically comprise a polynucleotidesequence at least substantially identical to the target gene linked to acomplementary polynucleotide sequence. The sequence and its complementare often connected through a linker sequence that allows thetranscribed RNA molecule to fold over such that the two sequenceshybridize to each other.

RNAi (e.g., siRNA, miRNA) appears to function by base-pairing tocomplementary RNA or DNA target sequences. When bound to RNA, theinhibitory RNA molecules trigger either RNA cleavage or translationalinhibition of the target sequence. When bound to DNA target sequences,it is thought that inhibitory RNAs can mediate DNA methylation of thetarget sequence. The consequence of these events, regardless of thespecific mechanism, is that gene expression is inhibited.

MicroRNAs (miRNAs) are noncoding RNAs of about 19 to about 24nucleotides in length that are processed from longer precursortranscripts that form stable hairpin structures.

In addition, antisense technology can be conveniently used. Toaccomplish this, a nucleic acid segment at least substantially identicalto the desired gene is cloned and operably linked to a promoter suchthat the antisense strand of RNA will be transcribed. The expressioncassette is then transformed into a plant and the antisense strand ofRNA is produced. In plant cells, it has been suggested that antisenseRNA inhibits gene expression by preventing the accumulation of mRNAwhich encodes the protein of interest.

Another method of suppression is sense suppression. Introduction ofexpression cassettes in which a nucleic acid is configured in the senseorientation with respect to the promoter has been shown to be aneffective means by which to block the transcription of target genes.

For these techniques, the introduced sequence in the expression cassetteneed not have absolute identity to the target gene. In addition, thesequence need not be full length, relative to either the primarytranscription product or fully processed mRNA. One of skill in the artwill also recognize that using these technologies families of genes canbe suppressed with a transcript. For instance, if a transcript isdesigned to have a sequence that is conserved among a family of genes,then multiple members of a gene family can be suppressed. Conversely, ifthe goal is to only suppress one member of a homologous gene family,then the transcript should be targeted to sequences with the mostvariance between family members.

Gene expression can also be inactivated using recombinant DNA techniquesby transforming plant cells with constructs comprising transposons orT-DNA sequences. Mutants prepared by these methods are identifiedaccording to standard techniques. For instance, mutants can be detectedby PCR or by detecting the presence or absence of CV mRNA, e.g., bynorthern blots or reverse transcriptase PCR (RT-PCR).

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of embryo-specific genes. It is possible to design ribozymesthat specifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA cleaving activity upon them,thereby increasing the activity of the constructs. The design and use oftarget RNA-specific ribozymes is well known.

Plant Transformation

Once an expression cassette comprising a CV polynucleotide of thepresent invention has been constructed, any technique known to thoseskilled in the art may be used to introduce the expression cassette intoa plant.

Methods for transformation and regeneration of plants are well known inthe art, and the selection of the most appropriate transformationtechnique for a particular embodiment of the invention may be determinedby the practitioner. Suitable methods may include, but are not limitedto: electroporation of plant protoplasts; liposome-mediatedtransformation; polyethylene glycol (PEG) mediated transformation;transformation using viruses; micro-injection of plant cells;micro-projectile bombardment of plant cells; vacuum infiltration; andAgrobacterium tumeficiens mediated transformation. Transformation meansintroducing a nucleotide sequence in a plant in a manner to cause stableor transient expression of the sequence.

Following transformation, cells or plants can be selected using adominant selectable marker incorporated into the transformation vector.Typically, such a marker will confer antibiotic or herbicide resistanceon the transformed cells or plants, and selection of transformants canbe accomplished by exposing the cells or plants to appropriateconcentrations of the antibiotic or herbicide.

Transformed cells may be regenerated into plants in accordance withtechniques well known to those of skill in the art. The regeneratedplants may then be grown, and crossed with the same or different plantvarieties using traditional breeding techniques to produce desiredplants. Two or more generations may be grown to ensure that the desiredphenotype (e.g., stress tolerance) is stably maintained and inheritedand then seeds harvested to ensure the desired phenotype or otherproperty has been achieved.

In some embodiments, the methods of the invention include a step ofselecting plants with the desired traits. The plants made by the methodsof the invention can be screened by well-known techniques, depending onthe desired trait. The determination that a plant modified according toa method of the invention has enhanced nutrient assimilation in desiredtissues (e.g., fruit or seeds) can be made by comparing yield of amodified plant with yield of a control (reference) plant that has notbeen modified. A plant of the invention may show increase in yield of atleast about 110%, preferably at least about 150%, more preferably atleast about 200%, as compared to a corresponding unmodified referenceplant.

Plants showing enhanced stress tolerance can be selected according tothe particular stress condition. For example, a plant having increasedsalt tolerance can be identified by growing the plant on a medium suchas soil that contains salt at a level more than about 100% of the amountof salt in the medium on which the corresponding reference plant iscapable of growing. Advantageously, a plant treated according to amethod of the invention can grow on a medium or soil containing salt ata level of at least about 110%, preferably at least about 150%, morepreferably at least about 200%, and optimally at least about 400% of thelevel of salt in the medium or soil on which a corresponding referenceplant can grow.

Drought-tolerance can be determined by any of a number of standardmeasures including turgor pressure, growth, yield, and the like. Forexample, a plant having increased tolerance to drought can be identifiedby growing the plant under conditions in which less than the optimalamount of water is provided to the plant through precipitation and/orirrigation. Particularly, a plant having increased tolerance to droughtcan be identified by growing the plant on a medium such as soil thatcontains less water than the medium on which the corresponding referenceplant is capable of growing. Advantageously, a plant treated accordingto a method of the invention can grow on a medium or soil containingwater at a level of less than about 90%, preferably less than about 80%,more preferably less than about 50%, and optimally less than about 20%of the amount of water in the medium or soil on which a correspondingreference plant can grow. Alternatively, a plant having increasedtolerance to drought can be identified by its ability to recover fromdrought (little or no applied water) when rehydration is provided aftera period of drought. Advantageously, a plant treated according to amethod of the invention can recover when rehydration is provided after aperiod of at least 3 days drought, at least 5 days drought, preferablyat least 7 days drought, more preferably at least about 10 days drought,and optimally at least about 18 days drought.

Water use efficiency can be determined by evaluating the amount of drybiomass that a plant accumulates (which can be vegetative, reproductive,or both, depending on the yield component(s) of interest) per unit wateravailable to the plant. A plant having enhanced water use efficiencywill have a greater amount of dry biomass accumulation per unit wateravailable than the corresponding reference plant grown under the sameconditions. Water use efficiency at the leaf or plant scale refers tothe ratio between the net CO2 assimilation rate and the transpirationrate, usually measured over a period of seconds or minutes. A plant withenhanced water use efficiency will have higher yields (such as 1-5%,5-10%, 10-15% higher) under restricted water conditions compared to thecorresponding reference plant grown under the same conditions.

Heat tolerance can be determined by evaluating the amount of dry biomassthat a plant accumulates (which can be vegetative, reproductive, orboth, depending on the yield component(s) of interest) relative toincreasing temperatures. A plant having enhanced heat tolerance willhave higher yields (such as 1-5%, 5-10%, 10-15% higher) under increasedtemperature conditions (such as 1° C., 2° C., 3° C., 4° C., etc.)compared to the corresponding reference plant grown under the sameconditions.

Once the appropriate selections are made, the process is repeated. Theprocess of backcrossing to the recurrent parent and selecting for thedesired trait is repeated for a number of generations. The lastbackcross generation can then be selfed in order to provide forhomozygous pure breeding progeny.

Methods of Making Nontransgenic Plants

A number of means are available for knocking out or inactivating anendogenous CV gene without using recombinant techniques. Thus, theplants produced by these methods are not transgenic.

Methods for introducing genetic mutations into plant genes and selectingplants with desired traits are well known. For instance, seeds or otherplant material can be treated with a mutagenic chemical substance,according to standard techniques. Such chemical substances include,diethyl sulfate, ethylene imine, ethyl methanesulfonate (EMS) andN-nitroso-N-ethylurea. Alternatively, ionizing radiation from sourcessuch as, X-rays or gamma rays can be used.

The resulting mutant plants can then be selected for mutations in the CVgene by a number of methods. For example, TILLING (Targeting InducedLocal Lesions IN Genomics) can be used to select plants in which the CVgene is knocked out. (See, e.g., McCallum et al., (2000), Plant Physiol123:439-442; McCallum et al., (2000) Nat Biotechnol 18:455-457; and,Colbert et al., (2001) Plant Physiol 126:480-484.

TILLING combines introduction of high density point mutations with rapiddetection of the mutations. Any mutagen (e.g., EMS) can be used tomutagenize plant seed. The mutant plants are then self-fertilized andthe resultant plants are then screened for mutation in the CV geneand/or for specific phenotypes. In a typical procedure, DNA frommutagenized plants is pooled and mutations in a CV gene are detected bydetection of heteroduplex formation. To do this, the CV gene in eachpooled sample is amplified (e.g., by PCR) and then denatured andannealed to allow formation of heteroduplexes, which indicate thepresence of one or more point mutations in the CV gene. Heteroduplexescan be identified by Denaturing High Performance Liquid Chromatography(DPHPLC). Typically, chromatography is performed while heating the DNA.Heteroduplexes have lower thermal stability, resulting in fastermovement in the chromatography column. As a result, the pools that carrya mutation in a CV gene are identified. Individual DNA from plants thatmake up the selected pooled population can then be identified andsequenced.

Other methods for detecting mutations in a CV gene include constantdenaturant capillary electrophoresis and single-stranded conformationalpolymorphism. Heteroduplexes can also be detected by using mismatchrepair enzymology. See Colbert et al., (2001) Plant Physiol 126:480-484.

Mutations in CV genes can also be introduced in a site-specific mannerby artificial zinc finger nuclease (ZFN), TAL effector (TALEN) orCRISPR/Cas technologies as known in the art. CRISPR (Clustered RegularlyInterspaced Short Palindromic Repeats)/Cas (CRISPR-associated) systems,are adaptive defense systems in prokaryotic organisms that cleaveforeign DNA. In the typical system, a Cas9 RNA-guided endonuclease isguided to a desired site in the genome using customizable small RNAsthat target sequence-specific single- or double-stranded DNA sequences.The CRISP/Cas system has been used to induce site specific mutations inplants (see Miao et al. (2013) Cell Research 23:1233-1236).

The non-transgenic plants made by any of the above methods can beselected for the desired stress tolerance trait (e.g., droughttolerance, salt tolerance, and the like) using any of the selectionmethods described above.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Methods Plant Materials and Growth Conditions

Arabidopsis thaliana (Col-0) plants were grown in a growth chamber at23° C. under a 16 h light/8 h dark regime. MS/2 media (0.5% sucrose,pH5.7) were used for plate-grown plants. Transgenic Arabidopsis plantsexpressing stroma-targeted DsRed (CT-DsRed) were generated as describedpreviously (Ishida et al. 2008, Plant Physiol 148: 142-155). Thegeneration of transgenic plant GTP-ATG8a (Thompson et al. 2005, PlantPhysiology 138: 2097-2110), the vacuole marker line VAMP711-RFP (Uemuraet al. 2004, Cell Structure And Function 29: 49-65), and theplastoglobule marker line PGL34-YFP (Vidi et al. 2007, BMC Biotechnol 7)were performed as described previously.

All the constructs in this study were generated using the Gateway system(Invitrogen, Grand Island, N.Y.). cDNA of AtCV (At2G25625) was amplifiedfrom mature leaf cDNA of Col-0. The 3′-terminus of AtCV gene was fusedwith GFP by fusion PCR and a linker (GGAAGGAA) was introduced betweenAtCV and GFP. The single AtCV gene and the fusion fragment(AtCV-linker-GFP) were both cloned into pDONR207 by BP reactions. ThepDONR207-AtCV was recombined via LR reactions into destination vectors:pEarley-Gate 101 (Earley et al. 2006 Plant Journal 45: 616-629) for YFPfusion (AtCV-YFP), pB7RWG2 (https://gateway.psb.ugent.be/search) for RFPfusion (AtCV-RFP), and a chemicalinducible system pBAV 154 (Vinatzer etal. 2006, Mol Microbiol 62: 26-44) for stable transformation(DEX:AtCVHA). The pDONR207-AtCV-linker-GFP was recombined intopEarley-Gate 100 for transient expression (AtCV-GFP) andchemical-inducible system pBAV 154 for stable transformation(DEX:AtCV-GFP), respectively. An artificial miRNA(TTACACGTAATGAACTTCCAG, SEQ ID NO: 47) targeting AtCV (amiR-AtCV) wasdesigned with WMD3 (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi) andcloned (Schwab et al. 2006, Plant Cell 18: 1121-1133) into pEarley-Gate100 for stable transformation. Using the same strategy, the genes ofAtPsbO1 (At5G66570), AtCYP20-2 (At5G13120), and FtsH1 (At1G50250) werefused with CFP first and then recombined into pEarley-Gate 100 to obtainconstructs PsbO1-CFP, CYP20-2-CFP, and FtsH1-CFP, respectively.Meanwhile, the deletion mutagenesis of chloroplast transit signalpeptide (Ml-L22) and C-terminus conserved domain (R92-V 152) wasperformed by PCR and the mutation fragment were fused with GFP andrecombined into pEarley-Gate 100 to generate constructs AtCVΔCGFP andAtCVΔN-GFP. The transient expressions were performed in cotyledons ofCol-0 young seedlings, as described previously (Marion et al. 2008, ThePlant Journal: For Cell And Molecular Biology 56: 169-179). The stabletransformation was performed according to the floral dipping method(Clough and Bent 1998, Plant Journal 16: 735-743).

RNA Extraction and Quantitative RT-PCR

For assessing senescence-induced AtCV expression, total RNA wasextracted from cassette leaf 7 of Col-0 plants growing in soil under 16h light/8 h dark. For testing abiotic stress-induced AtCV expression,total RNA was extracted from all the leaves of 10-dayold seedlingsgrowing without (control) or with 100 mM NaCl or 2 μM methyl viologen(MV) for 2 days. For assessing artificial miRNA silencing of AtCV, thecassette leaf 7 from 30-day-old plants of Col-0 and amiR-AtCV lines wereused for total RNA extraction. Total RNA was extracted by using RNeasyMini Kit (Qiagen, Redwood City, Calif.) with three biologicalreplicates. First-strand cDNA was synthesized from 1 μg of total RNAwith QuantiTech reverse transcription kit (Qiagen). qPCR was performedon the StepOnePlus (Applied Biosystems, Grand Island, N.Y.) using SYBRGREEN (Bio-Rad, Hercules, Calif.). The 2-ΔΔCT method (Livak andSchmittgen 2001, Methods 25: 402-408) was used to normalize anddetermine the mRNA level relative to an internal reference gene,TIP41-like family protein.

Fluorescence and Confocal Microscopy

Fluorescence microscopy was performed using an Inverted Zeiss LSM 710confocal laser scanning microscope (Carl Zeiss AG) equipped with a ×40water immersion objective. For GFP, the excitation wavelength was 488 nmand emission was 500-530 nm, CFP (440 nm/460-490 nm), YFP (514nm/525-552 nm), DsRed (543 nm/575-625 nm), lysotracker Red (561nm/570-600 nm), RFP (561 nm/600-660 nm), and Chlorophyll (633 nm/650-720nm). To avoid crosstalk between the fluorescence channels, sequentialscanning was used when necessary. Images were processed by ImageJ(rsbweb.nih.gov/ij/) and assembled by Photoshop software (Adobe).

Immunolabeling Transmission Electron Microscopy

The 10-day-old seedlings of DEX:AtCV-GFP transgenic plants and Col-0were cultured in liquid MS/2 media containing 10 μM DEX for 20 h. Thecotyledons were observed by confocal microscope and the tissues withhigh expression of AtCV-GFP were fixed in paraformaldehyde (2%) andglutaraldehyde (2.5%) as previously described (Shipman and Inoue 2009,Febs Letters 583: 938-942). Immunolabeling was performed on ultrathinsections on Formva-coated grids using anti-GFP antibody (NovusBiologicals, Littleton, Co) and goat anti-rabbit secondary antibodyconjugated with 10 nm gold (Brithish BioCell International Ltd, Cardiff,South Glamorgan, United Kingdom). All the grids were stained with uranylacetate and lead citrate before being observed on a Phillips CM120Biotwin. Images were taken with a Gatan MegaScan digital camera (model794/20). For the double immunolabeling experiments, leaf sections fromthe transgenic line DEX:AtCV-HA (DEX-3) were blotted with anti-HAantibody (from mouse) and anti-PsbO (from rabbit), then treatedsubsequently with 5 nm gold-conjugated goat anti-mouse IgG and 20 nmgold-conjugated goat anti-rabbit IgG for 1 h. The grids were stainedwith uranyl acetate and lead citrate for observation.

Immunoblotting Analyses

Plant leaf tissues were weighed, frozen in liquid N2, and ground inthree volumes of 2× Laemmli sample buffer. Total proteins were separatedby SDS-PAGE and transferred to PVDF membrane (Bio-Rad, Hercules, Calif.,USA) and probed as previously described (Wang et al. 2011, Plant Cell23: 3412-3427). Monoclonal antibodies raised against HA tag werepurchased from Covance (Princeton, N.J., USA) (#MMS-101P). Antibodiesraised against PsbO (#A505092), sucrose phosphate synthase/SPS(#AS03035A), PsaB (#AS06166A), PsbA/D1 (#A501016), GS1/GS2 or GLN1/GLN2(AS08295), and Lhcb2 (#AS01003) were obtained from Agrisera (Vannas,Sweden). Horseradish peroxidase-conjugated secondary antibodies werepurchased from Santa Crus Biotechnology (Dallas, Tex., USA).

Immunoprecipitation and Liquid Chromatography-Tandem Mass Spectrometry(LCMS/MS)

Four-day-old seedlings of Col-0 and transgenic plants DEX:AtCV-HA-3(DEX-3) were cultured in MS/2 media containing 1004 DEX for 4 days andthen kept in the dark for additional 2 days. The shoots of plants wereharvested, ground in liquid N2 and incubated at 4° C. for 3 h with lysisbuffer provided by μMACS HA Isolation Kit (Miltenyl Biotec, San Diego,Calif., USA), containing Protease Inhibitor Cocktail (Sigma-Aldrich, St.Louis, Mo., USA). Co-immunoprecipitation was performed using anti-HAmagnetic beads from μMACS HA Isolation Kit (Miltenyl Biotec, San Diego,Calif., USA) and incubating the cell lysis with beads at 4° C. for 2 h.LC-MS/MS analysis was performed in Genome Center of UniversityCalifornia, Davis, as described previously (Shipman-Roston et al. 2010,Plant Physiol 152: 1297-1308). Scaffold (version Scaffold 3) was used tovalidate MS/MS-based peptide and protein identification. Peptideidentifications were accepted if they could be established at >80.0%probability. Protein identifications were accepted if they could beestablished at >95.0% probability and contained at least threeidentified peptides.

For immunoprecipitation of the PsbO protein, the seedlings of Col-0,transgenic plant DEX:AtCV-HA-3 and DEX::AtCVΔC-HA were treated with DEXby the above-mentioned procedures. Cell lysis was incubated withanti-PsbO antibody and magnetic Dynabeads Protein A (Life Technologies™,Grand Island, N.Y., USA) for 2 h at 4.0 and immunoprecipitated sampleswere checked by immunoblotting with anti-PsbO and anti-HA antibody,respectively.

Bimolecular Fluorescence Complementation

The four vectors pDEST-GWVYNE, pDEST-VYNE(R)GW, pDEST-GWSCYCE, andpDESTSCYCE(R)GW from the GATEWAY-based BiFC vector systems (Gehl et al.2009, Mol Plant 2: 1051-1058) were employed to fuse AtCV, PsbO1 andSGR1(At4G22920) with N-terminus of yellow fluorescent protein Venus(VenusN) and C-terminus of super cyan fluorescent protein (SCFPC),respectively, to obtain the constructs AtCV-SCFPC, VenusN-AtCV,PsbO1-VenusN, SCFPC-PsbO1, SGR1-VenusN, and AtCVΔC-SCFPC. All theconstructs were introduced into A. tumefaciens strain GV3101. Thetransient expression was performed in cotyledons of Col-0 youngseedlings, as described previously (Marion et al. 2008, The PlantJournal: For Cell And Molecular Biology 56: 169-179).

GUS Staining and Chlorophyll Measurement

For GUS staining, the whole seedlings were submerged in standard X-GlcAsolution (50 mM sodium phosphate buffer pH7.0, 10 mM EDTA, 0.1% TritonX-100, and 0.5 mg/mL X-GlcA) and vacuum infiltrated for 5 min. Incubateat 37° C. for 16 h to develop blue color, as described previously(Jefferson et al. 1987, Plant Journal 52: 197-209).

For chlorophyll measurements, the leaves were weighted and ground inliquid N2. The chlorophyll was extracted in 80% acetone and theabsorbance (A) at 663 nm and 645 nm was measured using spectrophotometry(DU-640, Beckman Coulter, Brea, Calif., USA). Total chlorophyll contentswere calculated as described elsewhere (Porra 2002, Photosynth Res 73:149-156).

Results AtCV Expression was Activated by Abiotic Stress and Senescencebut Suppressed by Cytokinin

During abiotic stress, the breakdown of the plant photosyntheticmachinery is a major factor in the reduction of CO2-assimilation byplants (Tambussi et al. 2000, Physiologia Plantarum 108: 398-404). Ithas been shown previously that the cytokinin-dependent inhibition ofdrought-induced senescence resulted in sustained photosynthetic activityduring the stress episode and enhanced tolerance to water deficit(Rivero et al. 2007, Proceedings of the National Academy of Sciences ofthe United States of America 104: 19631-19636; Rivero et al. 2009, PlantPhysiology 150: 1530-1540; Rivero et al. 2010, Plant and Cell Physiology51: 1929-1941). The expression of isopentenyl synthase (IPT), encoding akey enzyme in cytokinin synthesis, under the control of a maturation-and stress-induced promoter (pSARK) leads to the protection of thephotosynthetic apparatus and enhanced chloroplast stability (Rivero etal. 2010; Reguera et al. 2013, Plant Physiology 163: 1609-1622). UsingDNA microarrays, we analyzed RNA expression patterns in wild-type andtransgenic pSARK::IPT rice plants during water deficit (Peleg et al.2011, Plant Biotechnology Journal 9: 747-758; Reguera et al. 2013, PlantPhysiology 163: 1609-1622). The expression of one gene encoding achloroplast protein with unknown function (LOC_Os05g49940) was activatedby stress in the wildtype plants but not in the transgenic pSARK:IPTplants (Peleg et al., 2011, Plant Biotechnology Journal 9: 747-758).

The Arabidopsis genome contains At2g25625, a gene homologue toLOC_Os05g49940, whose function remains to be characterized. The publicmicroarray database (Winter et al. 2007, Plos One 2: e718) indicatedthat At2g25625 expression was hardly detectable in young tissues, butits expression was greatly induced by abiotic stress and senescence whena massive chloroplast degradation occurred (Hortensteiner 2006, AnnualReview of Plant Biology 57: 55-77; Martinez et al. 2008, Annual ReviewOf Plant Biology 61: 443-462). Therefore, we surmised that the genecould play role(s) in chloroplast destabilization.

The gene in Arabidopsis (At2G25625) was cloned and termed AtCV(Chloroplast Vesiculation) due to the subcellular localization of theencoded protein and its functions as revealed in this study. Asindicated by quantitative RT-PCR assays (FIGS. 1A and 1B), AtCVexpression was activated by senescence and abiotic stresses such as saltstress and methyl viologen (MV)-induced oxidative stress. AtCVexpression was significantly down-regulated by 3 h treatment withcytokinin, a phytohormone delaying senescence (Gan and Amasino 1995,Science 270: 1986-1988). To study the tissue specific expression ofAtCV, we cloned the AtCV gene's native promoter, consisting of a 2 kbupstream region before the start codon, and used it to drive thereporter gene β-glucuronidase (GUS). The GUS staining assays oftransgenic plants ProAtCV:GUS suggested that AtCV gene was expressedstrongly in senescent and mature leaves but not in young leaves of40-day-old plant. In leaves from 21-day-old seedlings, AtCV expressionwas hardly detectable but its expression was substantially enhanced bysalt stress treatment.

AtCV Targets Chloroplasts and Induces the Vesicle Formation inChloroplasts.

AtCV is predicted to contain a chloroplast transit signal peptide at theN terminus by the ChloroP 1.1 Server(http://www.cbs.dtu.dk/services/ChloroP/). In order to assess AtCVsubcellular localization, we fused the enhanced green fluorescenceprotein (GFP) to the C-terminus of AtCV. The fusion gene AtCV-GFP wastransiently expressed in cotyledons of Arabidopsis plants constitutivelyexpressing stroma-targeted DsRed (CT-DsRed) (Ishida et al. 2008). Theconfocal microscopy observations indicated that AtCV-GFP localized inchloroplasts and concentrated in some vesicle-like spots. TheAtCV-containing vesicles (CCVs) also aggregated outside of thechloroplast in some unknown compartments that included thestroma-targeted DsRed but not chlorophyll. Interestingly, AtCV-GFPlocalized in both the cytosol and chloroplasts in epidermal cells. Theexpression of GFP alone resulted in a green fluorescence signal notassociated with chloroplasts. In addition, the movement of CCV departingfrom chloroplasts was captured by timelapse observation of confocalmicroscope.

The chloroplast localization of AtCV was further assessed byimmunolabeling using antibodies raised against GFP. The immunolabeledgold particles were mostly associated with thylakoids or envelopemembranes rather than stroma before the formation of vesicles. AtCV'smembrane association can be explained by its predicted transmembranedomain (aramemnon.botanik.uni-koeln.de). In some AtCV-labeledchloroplasts, the envelope membrane lost integrity and thylakoidmembranes appeared swelled and unstacked. CCVs were observed attached tothe envelope membrane of disassembled chloroplasts or protruding fromthe unstacked thylakoid membranes. The detection of GFP byimmune-labelling TEM in cotyledon mesophyll cells of DEX:AtCV-GFPtransgenic plants showed that 87% of the gold particles were localizedin chloroplasts and CCVs. In addition, we used leaf sections fromtransgenic plants DEX:AtCV-HA (DEX-3) for the doubleimmunolabeling withanti-HA and anti-PsbO antibodies. The results showed that the CCVs thatare close to, but not associated with, broken chloroplasts could also belabeled by antibodies raised against PsbO, a subunit of photosystem IIcomplex localized in thylakoid membrane. Moreover, CCVs also containedTic20-II, a protein from chloroplast inner envelope membranes(Machettira et al. 2011, Plant Mol Biol 77: 381-390). These resultssuggested that CCVs generated from chloroplast membranes that weredisrupted by AtCV. These vesicles and disrupted chloroplast structureswere not seen in cotyledons from wild type seedlings.

AtCV-Containing Vesicles were Mobilized to the Vacuole Through a PathwayIndependent of Autophagy and SAVs

The role of autophagy in the mobilization of Rubisco and stroma proteinsto the vacuole is well established (Ohsumi 2001, Nature ReviewsMolecular Cell Biology 2: 211-216; Ishida et al. 2008, Plant Physiol148: 142-155; Bassham 2009; Wada et al. 2009). During autophagy,cytosolic components and intact or partially broken organelles areengulfed in membrane-bound vesicles, called autophagosomes, that deliverthe vesicle contents to the vacuole for degradation. We transientlyexpressed the AtCV-RFP fusion gene in cotyledons from transgenic plantsexpressing the autophagic marker GFP-ATG8a (Thompson et al. 2005, PlantPhysiology 138: 2097-2110). The red fluorescence of AtCV-RFP did notoverlap with the green fluorescence of GFP-ATG8a. Moreover, whenAtCV-GFP was expressed in autophagy-defective mutants atg5-1 (Ishida etal. 2008), CCVs were observed both inside and outside of thechloroplasts, further suggesting that the formation and trafficking ofCCVs were independent of autophagy.

During senescence, the formation of small acidic senescence associatedvacuoles (SAV) aid in the degradation of chloroplast proteins. SAVs areformed through a pathway that is independent of autophagy (Otegui et al.2005; Martinez et al. 2008 Plant Journal 56: 196-206; Carrion et al.2013, J Exp Bot 64: 4967-4980). To rule out a possible relationshipbetween CCVs and SAVs, we attempted staining cotyledons from plantsexpressing AtCV-GFP with Lysotracker Red, a fluorescent dye that stainsacidic lytic vesicles including SAVs (Otegui et al., 2005, Plant Journal41: 831-844). The lack of CCV staining by Lysotracker Red, indicatedthat CCV's milieu differed from that of SAVs. In addition, the transientco-expression of SAG12-RFP along with AtCV-GFP in cotyledon cells showedthat the SAV marker SAG12-RFP did not colocalized with AtCV-GFP.

A Dexamethasone-(DEX)-induced promoter was used to express AtCV-GFP.DEX:AtCV-GFP stably transformed plants were treated with DEX and GFPfluorescence was monitored. Six hours after DEX treatment, AtCV-GFP wasseen decorating mesophyll cell chloroplasts and stromules (stroma-filledtubules; Hanson and Sattarzadeh, 2008 Plant, Cell and Environment 31:646-657, and references therein) extending from the chloroplasts.Eighteen hours following DEX treatment, the CCVs moved out from thechloroplast along with the stroma-targeted CT-DsRed. These observationswere consistent with AtCV transient expression results. Similar resultswere observed in DEX-induced expression of AtCV-GFP in true leaf cells,and in cotyledon and hypocotyl cells. CCVs also could carry CT-DsRed outof chloroplasts and aggregate in cytosols of mesophyll cells of trueleaves. To exclude the possibility that CCVs were produced at the ER,DEX:AtCV-GFP transgenic plants were treated with DEX for 17 hours andwith Concanamycin A, an inhibitor of intracellular vesicle trafficking(Dettmer et al. 2006, Plant Cell 18: 715-730) for an additional hour.Concanamycin A treatment inhibited the release of CCVs from chloroplastssince the CCVs appeared adhered to the chloroplasts after treatment.

To assess whether CCVs were eventually transported to the vacuole, theAtCV-GFP was transiently expressed in stable report lines of Rab2a-RFP,a prevacuolar compartment rab5 GTPase Rha1 (Foresti et al. 2010, PlantCell 22: 3992-4008) and VAMP711-RFP, a tonoplast R-SNARE (Uemura et al.2004, Cell Structure And Function 29: 49-65). Our results showed thatAtCV-GFP overlapped with RabF2a-RFP and VAMP711-RFP in hypocotyls cells3 days after transient expression, supporting the mobilization of CCVsto the central vacuole.

AtCV Overexpression Leads to Chloroplast Degradation

Attempts to overexpress AtCV under the control of the CaMV35Sconstitutive promoter were not successful, suggesting that the high AtCVexpression could be lethal. We used an alternative approach utilizing achemically-inducible expression system to drive the expression of theAtCV-HA fusion gene. The phenotypical analysis of three independentstable lines DEX-1, DEX-2, and DEX-3 showed that DEX-induced AtCVexpression resulted in leaves chlorosis and growth retardation. The leafchlorophyll contents under DEX treatment decreased as compared with thatof untreated transgenic and wild-type plants. Western blot analysesrevealed that the PSI complex subunit PsaB, PSII subunits (PsbO1 and D1)and stromal protein glutamine synthase 2 (GS2) were degraded inDEX-treated plants. The levels of cytosolic sucrose phosphate synthase(SPS) remained unchanged upon DEX treatment, whereas the abundance ofcytosolic glutamine synthase 1 (GS1) increased, consistent with aprevious study showing the up-regulation of GS1 expression duringsenescence (Bernhard and Matile 1994 Plant Sci 98: 7-14). Oxidativestress, induced by the exposure of the plants to 0.3 μM methyl viologen,enhanced stress-induced chloroplast degradation in transgenic plantsexpressing AtCV. Overexpression of AtCV-GFP also induced the acceleratedsenescence phenotype under 50 mM NaCl. These results indicated that theover-expression of AtCV lead to premature senescence and chloroplastdegradation.

AtCV Silencing Lead to Delayed Chloroplast Degradation

An amiRNA targeting AtCV (amiR-AtCV) was designed using WMD3(wmd3.weigelworld.org/cgi-bin/webapp.cgi) (Schwab et al. 2006, PlantCell 18: 1121-1133) and its expression was driven by the CaMV35Spromoter. Three independent transgenic lines (amiR-AtCV1-3) wereselected and AtCV silencing was examined by quantitative RTPCR (FIG.1A). AtCV-silenced plants had no apparent developmental defects andtheir natural senescence was also indistinguishable from wild typeplants. However, when 20-day-old seedlings of wild-type and amiR-AtCVplants were treated with gradually-increasing NaCl concentrations (FIG.1B), the wild type plants displayed severe leaf senescence symptoms,while the amiR-AtCV plants remain green. Chlorophyll measurement ofwild-type cassette leaves indicated decrease in chlorophyll contentsduring the treatment, while salt stress-induced leaf senescence wasdelayed in AtCV-silenced plants (FIG. 1B). The degradation ofphotosystem I subunits (PsaB), photosystem II subunits (D1, PsbO1, andLhcb2) and stroma protein GS2 were clear in wild-type plants after 10days of salt treatment. In amiR-AtCV plants, the abundance of theabove-mentioned chloroplast proteins only decreased slightly after 16days of salt treatments. These results demonstrated that silencing AtCVinhibited the salt stress-induced degradation of chloroplast proteins.In addition, the chloroplast degradation caused by MV-induced oxidativestress was also delayed in amiR-AtCV lines. Moreover, their survivalrates increased significantly after 14-day drought treatment (FIG. 2).These results indicated that silencing AtCV increased chloroplaststability and prevented abiotic-stress-induced senescence.

The C-Terminal Domain of AtCV is Important for ChloroplastDestabilization and the Formation of CCVs.

A search of sequences similar to AtCV in the public genome databasesshowed the presence of AtCV homologs in all plant species sequenced sofar. These genes contain a unique highly conserved domain at theC-terminus of the encoded proteins. Without the conserved C terminusdomain, AtCVΔC-GFP still localized at the chloroplasts but hardlyproduced vesicles. Moreover, the DEX-induced expression of AtCVΔC-GFPproduced some leaf senescence and partial chloroplast degradation.Nonetheless, the destabilizing functions of AtCVΔCGFP were substantiallyimpaired, as compared with the plants expressing the full-lengthAtCV-GFP, indicating a key role of the conserved C-terminus domain ofAtCV in chloroplast destabilization and the formation of CCVs.

AtCV Interacts with Photosystem II Subunit PsbO In Vivo

To elucidate mechanism(s) by which AtCV disrupts chloroplasts, weidentified AtCV potential interacting proteins usingco-immunoprecipitation (Co-IP) and subsequent identification ofinteractors by LC-MS/MS (Smaczniak et al. 2012, Nat Protoc 7:2144-2158). Antibodies raised against HA were conjugated to magneticbeads, and the beads were used to immunoprecipitate AtCV-HA and itsinteracting proteins from total protein extracts obtained fromDEX-treated transgenic plants expressing DEX:AtCV-HA (DEX-3 line).Protein extracts from wild type Col-0 plants were used as a control todetect proteins that bind nonspecifically to the anti-HA beads. Most ofimmunoprecipitated proteins were chloroplast proteins includingPhotosystem II (PSII) complex subunits, NAD(P)H Dehydrogenase subunits,thylakoid membrane-bound proteases, and a few stromal proteins. Thesimilarity between the peptide abundances of PSII subunits PsbO1, PsbO2and the bait protein AtCV and their localization and functions wouldindicate the interaction between AtCV and PsbO proteins. In order toconfirm this interaction, we used bimolecular fluorescencecomplementation (BiFC). The transient expression of both fusion genesAtCV-SCFPC and PsbO1-VenusN in cotyledons of wild type seedlingsresulted in BiFC fluorescence that was seen not only in the chloroplastsbut also in the CCVs, whereas the coexpression of AtCV-SCFPC andSGR1-VenusN failed to produce green fluorescence signals in threeindependent tests. These results indicated a direct interaction betweenAtCV and PsbO1 in vivo. Interestingly, the co-expression of theN-terminus fusion SCFPC-PsbO1 and VenusN-AtCV also induced fluorescence,which was not associated with chloroplasts, suggesting that theN-terminal fusion did not affect the interaction between AtCV and PsbO1,but misled proteins to other location (perhaps cytosol) rather thanchloroplast because of the disruption of the N-terminus chloroplasttransit signal peptide of AtCV and PsbO1.

We also constructed another mutation AtCVΔC by deleting the AtCVC-terminus conserved domain. No fluorescence was detected betweenAtCVΔC-SCFPC and PsbO1-VenusN. These results indicated that theconserved C-terminus domain was required for the interaction betweenAtCV and PsbO1. This notion was further confirmed by the Co-IP resultsshowing that the full length AtCV, but not AtCVΔC, was immunopreciptatedby using anti-PsbO1 antibody. In addition, we transiently co-expressedAtCV-YFP together with PsbO1-CFP in wild type seedlings. In cellswithout AtCV-YFP, the PsbO1-CFP was distributed uniformly inchloroplasts. However, AtCV-YFP expression altered the localization ofPsbO1 and caused the concentration of PsbO1-CFP in the AtCV-containingvesicles. Collectively, these finding suggested that AtCV could disruptthe localization of PsbO1 in chloroplasts, possibly through directprotein-protein interaction.

In addition to the stroma-targeted DsRed and PSII subunit PsbO1, we alsoobserved two more thylakoid proteins “wrapped” in CCVs. The geneencoding the thylakoid lumen protein AtCYP20-2, an immunophilinassociated with the PSI/NDH supercomplex (Sirpio et al. 2009, FebsLetters 583: 2355-2358), was cloned and fused with CFP. The AtCYP20-CFPconstruct was co-expressed transiently with AtCV-GFP in cotyledons andconfocal microscopy observations clearly showed their co-localization.Also, the gene encoding the thylakoid membrane-bound FtsH protease wasfused to CFP and the AtFtsH1-CFP was co-expressed with AtCV-GFP. Ourresults showed that AtFtsH1-CFP and AtCV-GFP overlapped both inchloroplast and in CCVs released from the chloroplast. However, theplastoglobule marker protein plastoglobulin 34, PGL34-YFP (Vidi et al.2007, BMC Biotechnol 7), did not overlap with AtCV-RFP, suggesting thatthe plastoglobule turnover was independent of the AtCV-induceddegradation pathway.

DISCUSSION AtCV Regulates Stress-Induced Chloroplast Degradation Througha Pathway Independent of Autophagy and SAVs

Plants use different strategies to cope with environmental stress. The“escape” strategy involves the fast degradation of source tissues andthe accelerated development of sinks, contributing to a faster lifecycle and the production of seeds for the next generation (Levitt 1972,Annu Rev Plant Biol 58: 115-136). Chloroplasts contain large amounts ofproteins, and the fast and massive chloroplast degradation during stressis a key process that provides nutrients for relocation to developingorgans (Makino and Osmond 1991, Plant Physiol 96: 355-362). In thisstudy, we identified a gene AtCV encoding a protein that mediates theturnover of chloroplast proteins. Our results showed that silencing AtCVdelayed the stress-induced chloroplast degradation and leaf senescencewhile AtCV overexpression caused chloroplast degradation and prematureleaf senescence.

Previous studies revealed two extra-plastidic proteolytic processes,autophagy (Ishida et al. 2008, Plant Physiol 148: 142-155; Wada et al.2009, Plant Physiol 149: 885-893) and SAVs (Otegui et al. 2005; Martinezet al. 2008, Plant Journal 56: 196-206; Carrion et al. 2013), that areinvolved in the degradation of chloroplasts. However, little is knownabout the factors regulating intra-plastidic chloroplast degradation.Our results revealed a novel proteolytic pathway, which is independentof autophagy and SAVs, and is mediated by the formation ofAtCV-containing vesicles. AtCV expression is elicited by tissuesenescence or stress-induced senescence. After targeting thechloroplast, AtCV is able to induce the formation of vesicles inchloroplasts (CCVs) through a mechanism that is unclear so far. The CCVsare eventually released from the chloroplast to the cytosol carryingaway some “cargo” proteins from the chloroplast. In addition to stromalprotein, CCVs were shown to contain the thylakoid membrane proteinFtsH1, lumenal proteins PsbO1 and AtCYP20-2, and the inner envelopemembrane protein Tic20-II.

Based on the immunolabeling results, AtCV proteins are mostly associatedwith thylakoid membrane and envelope membrane before the formation ofCCVs, likely via its putative transmembrane domain. Confocal microscopyobservations also demonstrated the co-localization of AtCV and the innerenvelope membrane protein Tic20-II. Although the exact mechanism ofvesicle formation remains elusive, these results, together with ourobservations showing that the AtCV-induced vesicle formation werecoupled with the unstacking and swelling of the thylakoid membranes andthe disassembling of the chloroplast structure, support the notion thatthe CCVs can form directly from the chloroplast membranes disrupted byAtCV.

Autophagy induces the formation of Rubisco-containing bodies (RCBs) byengulfing the stromules protruding from chloroplasts and the chloroplastfunctions are still maintained (Ishida et al. 2008). As compared withautophagy-dependent degradation, AtCVmediated degradation appears to bemore destructive. AtCV-mediated chloroplast damage leads to leafsenescence, as observed during DEX-induced AtCV over-expression.

Interestingly, silencing AtCV did not delay the natural leaf senescence.A possible explanation of this phenomena is that other pathways, such asautophagy (Ishida et al. 2008, Plant Physiol 148: 142-155; Wada et al.2009, Plant Physiol 149: 885-893), SAVs (Otegui et al. 2005, PlantJournal 41: 831-844; Martinez et al. 2008, Plant Journal 56: 196-206;Carrion et al. 2013, J Exp Bot 64: 4967-4980) or SGR-mediatedchlorophyll degradation (Park et al. 2007, Plant Cell 19: 1649-1664; Renet al. 2007, Plant Physiology 144: 1429-1441; Hortensteiner 2009, TrendsPlant Sci 14: 155-162; Sakuraba et al. 2012), might be enhanced inAtCV-silenced plants for destabilizing chloroplasts and acceleratingsenescence. The possible interactions between the processes ofautophagy, SAVs and AtCV-dependent degradation are unknown and requirefurther investigation.

AtCV Mediates Chloroplast Destabilization and Vesiculation

Co-immunoprecipitation and subsequent analysis by LC-MS/MS revealedseveral proteins having potential interaction with AtCV. We confirmedthat AtCV targets PSII subunit PsbO1 in vivo by BiFC assays. AnotherPsbO gene product, PsbO2, that shares 91% similarity with PsbO1 in aminoacid sequence, was also immunoprecipitated by AtCV, suggesting theAtCV-PsbO2 interaction.

In spite of its role in stabilizing Manganese, PsbO is thought to play achaperone-like role in PSII assembly (Yamamoto 2001, Plant and CellPhysiology 42: 121-128, Plant and Cell Physiology 42: 121-128; Yamamotoet al. 2008, Photosynth Res 98: 589-608). Although PsbO1 and PsbO2functions are not completely redundant (Lundin et al. 2007, PlantJournal 49: 528-539), RNAi silencing of both genes (Yi et al. 2005,Journal of Biological Chemistry 280: 16170-16174) lead to a decreasedstability of PSII and the loss of some photosynthetic proteins,including CP47, CP43, D1, and even the PSI core protein PsaB, while thelight-harvesting complex II (LHC II) was stable in PsbO RNAi lines (Yiet al. 2005, Journal of Biological Chemistry 280: 16170-16174). In AtCVoverexpressing lines, D1 and PsaB were degraded while the stability ofLhcb2 was less affected as compared with other PSII proteins.Furthermore, CP43 and D1 were also immunoprecipitated by AtCV.Altogether, these results strongly suggested the functional interactionbetween AtCV and PsbO. AtCV targeted PsbO directly and might alter thestructure of PSII complex, removing PsbO, affecting PSII stability, andmaking core proteins (such as D1) very susceptible to thylakoidproteases. The proteases Deg (Kapri-Pardes et al. 2007, Plant Cell19:1039-1047) and FstH (Lindahl et al. 2000, Plant Cell 12: 419-431;Zaltsman et al. 2005, Plant Cell 17: 2782-2790; Shen et al. 2007, PlantJ 52: 309-321; Adam et al. 2011, Plant Cell 23: 3745-3760) have beenidentified to be responsible for the turnover of D1 protein.Interestingly, both DegP1 and FstH1 appeared to interact with AtCV andFstH1-CFP colocalized with AtCV-GFP in vivo. Taken together, theseresults suggest a mechanism by which AtCV might facilitate the approachof proteases to D1 protein after removing PsbO. AtCV-dependent removalof PsbO promotes PSII turnover and destabilizes chloroplasts. Inaddition, previous in-vitro studies revealed that the aggregation of D1and other subunits including CP43 occurred in the absence of PsbO (Henmiet al. 2003, Plant and

Cell Physiology 44: 451-456; Yamamoto et al. 2008, Photosynth Res 98:589-608). Thus, AtCV-induced elimination of PsbO could cause theaggregation between D1 and other PSII core proteins, and thisaggregation could signal for vesicle formation. Supporting this notion,it has been shown recently that the over-expression of triple geneblock3 (TGB3) of Alternanthera mosaic virus in Nicotinana benthamianacaused chloroplast vesiculation and veinal necrosis by interacting withthe host PsbO (Jang et al. 2013, Front Plant Sci 4). AtCV interacts withPsbO1 by a Cterminus domain which is highly conserved in the plantkingdom. The conserved domain appeared to be important for vesicleformation and chloroplast degradation. However, the deletion of theconserved domain did not completely eliminate chloroplast function.Several chloroplast proteins, in addition to PsbO, were alsoimmunoprecipitated by AtCV, suggesting that PsbO1 may not be the onlyprotein targeted by AtCV during the process of chloroplast degradation.

Increase Stress Tolerance Through Stabilizing the Chloroplast

Abiotic stress limits plant growth and productivity by disruptingphotosynthesis and inducing senescence. Emerging evidence suggested thatchloroplast stability plays a significant role in the tolerance ofplants to abiotic stress. Senescence and stress-induced synthesis ofcytokinin synthesis delayed the degradation of photosynthetic complexesin transgenic plants expressing PSARK:IPT plants that displayed enhanceddrought tolerance (Rivero et al. 2010, Plant and Cell Physiology 51:1929-1941). In addition, a wheat stay-green mutant (tasg1) displayed adelayed chlorophyll turnover and improved tolerance to drought becauseof the enhanced stability of thylakoid membranes (Tian et al. 2013, JExp Bot 64:1509-1520). The stable chloroplasts could also contribute tomaintain photorespiration which has been shown to increase the toleranceto abiotic stress by protecting the photosynthetic apparatus fromoxidative damage and optimizing photosynthesis (Rivero et al. 2009,Plant Physiology 150: 1530-1540; Voss et al. 2013, Plant Biology15:713-722). Here, we showed that silencing AtCV increased thechloroplast stability and prevented premature senescence under salt,oxidative and drought stress (FIG. 1). Our results would indicate thatAtCV acts as a scaffold targeting PSII proteins directly. Thus silencingAtCV may protect PSII functions, increasing plant tolerance to abioticstress. Moreover, AtCV-silenced plants displayed increased glutaminesynthase 2 stability. GS2 is a major enzyme for nitrogen assimilation,and GS2 overexpression lead to increased salt stress tolerance in rice(Hoshida et al. 2000, Plant MolBiol 43: 103-111).

In conclusion, our results provide evidence supporting a novel pathwayfor the degradation of thylakoid and stromal proteins that isindependent of autophagy (Ishida, 2008 Autophagy 4: 961-962) and SAVs(Otegui et al. 2005, Plant Journal 41: 831-844; Martinez et al. 2008,Plant Journal 56: 196-206; Carrion et al. 2013). While authophagy isresponsible for general cellular degradation, AtCV appears to be uniqueand specific for chloroplast degradation. From a biotechnologicalperspective, silencing of AtCV offers a suitable strategy for thegeneration of transgenic crops with increased tolerance to abioticstress.

Example 2

This example shows that rice lines expressing amiRNAs are resistant todrought stress. Transgenic rice plants were prepared according tostandard techniques using the amiRNAs described above.

Wild type and 3 transgenic rice lines expressing amiRNAs were grown inthe greenhouse under well watered conditions (black bars in FIG. 3) andsubjected to water deficit stress (water witheld for 6-8 days, gray barsin FIG. 3). Plants were re-watered and seeds were collected. Results inFIG. 3 show that although the transgenic plants displayed some yieldpenalty at well-watered conditions, they yielded significantly moreseeds after the stress event.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

INFORMAL SEQUENCE LISTING >AT2G25625 (Arabidopsis thaliana) SEQ ID NO: 1ATGGCAGGGAGAATAAGCTGCTGTCTAAATCTTCCTCCCCTAGATTCAAATTCTGCACAATCATTAGCTTCACTGCTCAAGACAACGTCAAAGATCTCTTGTAGGAGAACAGAAAATGAGACAGAGCCACGGAAAAACAAGTGTTCTTTTGTCTTGGGGGTGGCGGCAACTGTCGTAATCGGCGGGATTCAGATCAATGATGTTGCATCAGTGGAAGCTGCGGTTGTGAAATCGCCGGTAGAAGAGATGGCTGCGGGTGTGGTGCCGCCGCGGAGGTGGAGTGACAAGAGGACGTGTCCGCCTTGGCTTGAGAACTCGCTAGAGACCATCGTACCGGAGAATCTTCCTCGTCCGTCTGCTCATCGACGTTTGGAGTTAGCCGGATTAGCTAAGGGTGATGCACCGCCGGTCGGTGTGGTGATGACACGTGTCAACAGGGGTGGTTGTTTCTCCGTGTAA SEQ ID NO: 2MAGRISCCLNLPPLDSNSAQSLASLLKTTSKISCRRTENETEPRKNKCSFVLGVAATVVIGGIQINDVASVEAAVVKSPVEEMAAGVVPPRRWSDKRTCPPWLENSLETIVPENLPRPSAHRRLELAGLAKGDAPPVGVVMTRVNRGGCFSV<BD2G15690 (Brachypodium distachyon) SEQ ID NO: 3ATGCCGGTCTCCTCCGCCATCAGCTGCTGCCAGCAGCTCAGACCTCCGGGTCCACCTCCGGCGCCGAGAGAAGAAGGCAGCAGCAGCAGTAGTCGTATCCCGCTGTCGCGGCGCAGGGCGTGCTTTCTCGCGGCGGCGGCGGCGTGCGTGGTGGCCGTGGCGGCGGGAGGGGCCGGAGAAGCAGCGGCGATGGCGCGGGGCGCGCCGCACGAGCACGAGGCGGCGGCGGCGGTGCGCGTGCAGGCTGGGGCGGCGGCGAGGTGGAGCGAGCGCAGGCAGTGCCCGCCGTGGCGGGCGAATTCGCTGGAGAACGTAGTGCCGGAGAACCTGCCGCGCCCGTCGGCGCGGCGGAGGTACAACGGCGTCGGGGAGAGGGATCCCGCGCCGGCGCCAGCCGCGTCGCCTGAGGCCGTGCTCCCGTTCCTGGCGCTGCGCTCCGGCGGCATGGGCTGCTTCTCTCTCTAA SEQ ID NO: 4MPVSSAISCCQQLRPPGPPPAPREEGSSSSSRIPLSRRRACFLAAAAACVVAVAAGGAGEAAAMARGAPHEHEAAAAVRVQAGAAARWSERRQCPPWRANSLENVVPENLPRPSARRRYNGVGERDPAPAPAASPEAVLPFLALRSGGMGCFSL>CP00603G00010 (Carica papaya) SEQ ID NO: 5ATGGATATGGAGGATAGATTACAGAAGAAATGTGGAGATTTGAGCCTGATCTACTCCGGAAGACATGAGAGAGGTTTTTGGGCGATTGACACGAGGATTCCTGGTTTCCGGCGAATGCTCAGGAACAATGTGAAGAGGAGTCGTCTGGTTAAACGAAACGGTAGAGAGGGAGAGAGTGAAGATGATGGCGATGAGAGTAAGTTGCTGCCTAAACCTTCCTCCTCGAGAAAAACCTTCACTCCATCTCCCTCCTCCTGCCAACACGTTTTCTCAATTACCTTCAAGGAAAAAGGAAGAATGTTGGAGGAAGAAGAGTGCGGTGGCGATGGCGTGCTTGGTAATTGGATTGATACAGGTGGCAAATGGAGCGAAAAGAGAATGTGTCCGCCATGGCATGCTAACTCCCTTGAGACCGTCGTGCCGGAGAATCTCCCTCGGCCATCTGCTCGTCGGAGATGGCAGCTTGTTGCTTTCACCCCCAACCTTCGTCAGCCCCCACCCACATGTTTCTCTTTGTGA SEQ ID NO: 6MDMEDRLQKKCGDLSLIYSGRHERGFWAIDTRIPGFRRMLRNNVKRSRLVKRNGREGESEDDGDESKLLPKPSSSRKTFTPSPSSCQHVFSITFKEKGRMLEEEECGGDGVLGNWIDTGGKWSEKRMCPPWHANSLETVVPENLPRPSARRRWQLVAFTPNLRQPPPTCF SL>FV2G40830 (Fragaria vesca) SEQ ID NO: 7ATGGCCATGACAAGCTTCAGCTGCAGCCTTAACCAGCTGCCGCCTCCAGCTCAAAGCTTAGGCCCTTCTTCCCCTTCAAAGACGAATCAAGTACAACTTGCATGGAACAAAAGCGAGGGAGGATCATGGAGTAGCAGATGTGTTGTGGGCATGGCTTGTGTTATGGTTGGGTTGGAGATGGGTGGTTTGGTGAGTGGCCAAAGCCATGAAGCTATTGCTAAAGGTATGCCGCCGTTGGTGATGGAGTCAAGTGAGAAAGTTGCAAAGTGGAGTGACAAGAGAATGTGCCCAAAGTGGAGAGCCAATGAGCTGGAGACCATTGTGCCGGAGAATCTTCCGAGGCCGTCGGCTCACCGGAGATGGGAAATCGTCGGGTTTAATACTAGGGATGCTCCGGCGGTTAAGACGGTAGCTAGGAGGAGTAGCGGTGGTTGC TTTTCTATGTAASEQ ID NO: 8 MAMTSFSCSLNQLPPPAQSLGPSSPSKTNQVQLAWNKSEGGSWSSRCVVGMACVMVGLEMGGLVSGQSHEAIAKGMPPLVMESSEKVAKWSDKRMCPKWRANELETIVPENLPRPSAHRRWEIVGFNTRDAPAVKTVARRSSGGCFSM >GM04G07440 (Glycine max-1)SEQ ID NO: 9 ATGAGGACCACTTGCTTACTAAGCCTTCCCCCTCTTACTTCAAACCAACCCTCCAACGCTTCTTTCAACCCCGCAAAGCCACCTCAACTTTCATCGCAATGCGTTATGATGGGAGTGGCATCCATAATTGGACTAGAAATGTGCAATTTAGTGGCACTGGCCCACGAAGCAATTGAAATCACAACTATGCCAATTGGTAACCAAGTAAAAAGAACGTGCTCACCTTGGCAAGGCAACTCGCTCGAAACCATCATGCCGGAGAACCTTCCCCGGCCGTCGGCACGGCGGCGATACGAGGCTGTTCGTTCCTCCACCAAGACTGTGCCACCGTCCTCAGCCCCGATCATAGTCCAAAGCAACAAGGGCAGCTGCTTCTCCATGTG A SEQ ID NO: 10MRTTCLLSLPPLTSNQPSNASFNPAKPPQLSSQCVMMGVASIIGLEMCNLVALAHEAIEITTMPIGNQVKRTCSPWQGNSLETIMPENLPRPSARRRYEAVRSSTKTVPPSSAPIIV QSNKGSCFSM>GM06G07560 (Glycine max-2) SEQ ID NO: 11ATGAGGACCAGTTGCTTCCTAAGCCTTCCCCCTTTTACTTCAAACCAACCTTCCATTCCCCCAAAACATCCTCAACTTTCATCGGTGAAGAACGAAGCATGTTGGAAGAGGCAATGCGTTGTGATGGGAGTGGCATCCATTATTGGACTAGAAATGTGCAATTCAGTGGCAATGGCCCACGAAGCAATTGAAATCAAGACCATGCCATTTAGTAACCAAGTAGTATCAAATAGCAATTCTTACGGTGGCGCCAAATGGAGCGAGAAAAGAATGTGCCCACCTTGGCAAGGCAATTCGCTCGAAACGATCGTGCCGGAGAATCTTCCCCGGCCGTCGGCACGGCGGAGATACGAGGCTGTTCGTTCCTCCTCCAAGACTGCGCCGCCGCTCTCCGCCCCGATCATAGTCCAAAGCAACAAGGGCAGTTGCTTCTCCATG TGASEQ ID NO: 12 MRTSCFLSLPPFTSNQPSIPPKHPQLSSVKNEACWKRQCVVMGVASIIGLEMCNSVAMAHEAIEIKTMPFSNQVVSNSNSYGGAKWSEKRMCPPWQGNSLETIVPENLPRPSARRRYEAVRSSSKTAPPLSAPIIVQSNKGSCFSM >LJ0G026030 (Lotus japonicus)SEQ ID NO: 13 GCCAAATGGAGCCAGAAAAGGGCGTGTCCTCCTTGGCGAGGTAACGCTTTGGAAACCATCGTGCCGGAGAATCTTCCGCGGCCAGCGGCGCGGCGGAGATACGAGGCTGTTCGGTCAACCTCCAAGACGGCGCCGCCGCTCTCTGAAGCCTTCAAAATTAAATCCAACAGTTATAGTTGCTTCTCCATG SEQ ID NO: 14AKWSQKRACPPWRGNALETIVPENLPRPAARRRYEAVRSTSKTAPPLSEAFKIKSNS YSCFSM>MD00G178660 (Malus domestica-1) SEQ ID NO: 15ATGGCTTGTTTTAGAGGGTCACCATTGAGGTCTTTGTCATCTCTTTTAACCTTATGCAACCAAACCCACCTTCCTCTTTTGATATATACGATCCCTCTCCTTCTGTCATTGCTTAAATTCAGAGAGAACAGAGAGAGAGAGAGAGAGAAAGGGATGACTGTTACAAACTTCAATTGCTGCCTCAATCCGCCACCTTCAAATCAAAACCATGGTTCAAGCCCTTCTTTGCCCTTAAAGAAAAACCAAGCACTTGCATGGAACAAATATGCTCATGGATCATGGACTAATCGATGCGTTTTAGGTATGAGTTGCGCAATTGGATTGGAAATGGGAACCCTAGTAAGCAACCAAAACTATGAGGCCATTGCTAATGCTATGCCTTCGCCGTTGGAAATAGAAACATATAGTGATCAGAGGGTTGAAAAATGGAGTGACAAAAGAATATGCCCACAATGGAGCCCTAATTCACAAGAGACCATTGTGCCTGAAAATCTCCCAAGATCATCTGCTCAAAGGAGATGGGAAACAGTTGGTTTTTCTAACGAGGATGCTCCGGCGGTTCAAATGGTAGTTAGAAAAGGTGGCAACTGCTTTGCTATGTAG SEQ ID NO: 16MACFRGSPLRSLSSLLTLCNQTHLPLLIYTIPLLLSLLKFRENREREREKGMTVTNFNCCLNPPPSNQNHGSSPSLPLKKNQALAWNKYAHGSWTNRCVLGMSCAIGLEMGTLVSNQNYEAIANAMPSPLEIETYSDQRVEKWSDKRICPQWSPNSQETIVPENLPRSSAQRRWETVGFSNEDAPAVQMVVRKGGNCFAM >MD00G406410 (Malus domestica-2)SEQ ID NO: 17 ATGACTGTTACAAACTTCAATTGCTGCCTCAATCCGCCACCTTCAAATCAAAACCATGGTTCAAGCCCTTCTTTGCCCTTAAAGCAAAACCAAGCACTTGCATGGAACAAATATGCTCATGGATCATGGACTAATCGATGCGTTTTAGGTATGAGTTGCATCGCAATTGGATATGAAATGGGAACCCTAGTAAGCAACCAAAACTATGAGGCCATTGCTAATGCTATGCCTTCGCCGTTGGAAATAGAAACATCAAGTGATCAGAGGGTTGCAAAATGGAGTGACAAAAGAATGTGCCCACAATGGAGCCCTAATTCGCTAGAGACCATTGTGCCTGAAAATCTTCCAAGACCATCTGCTCAAATGAGATGGGAAACCGTTGGTTTTTCTGACAAGGATGCTCCGGTGGTTCAAATGGTAGTTAGAAAAGGTGGCAG CTGCTTTGCTATGTAGSEQ ID NO: 18 MTVTNFNCCLNPPPSNQNHGSSPSLPLKQNQALAWNKYAHGSWTNRCVLGMSCIAIGYEMGTLVSNQNYEAIANAMPSPLEIETSSDQRVAKWSDKRMCPQWSPNSLETIVPENLPRPSAQMRWETVGFSDKDAPVVQMVVRKGGSCFAM >MD14G005720 (Malus domestica-3)SEQ ID NO: 19 ATGGCTTGTTTTAGAGGGTCACCATTGAGGTCTTTGTCATCTCTTTTAACCTTATGCAACCAAACCCACCTTCCTCTTTTGATATATACGATCCCTCTCCTTCTGTCATTGCTTAAATTCAGAGAGAACAGAGAGAGAGAGAGAGAGAAAGGGATGACTGTTACAAACTTCAATTGCTGCCTCAATCCGCCACCTTCAAATCAAAACCATGGTTCAAGCCCTTCTTTGCCCTTAAAGAAAAACCAAGCACTTGCATGGAACAAATATGCTCATGGATCATGGACTAATCGATGCGTTTTAGGTATGAGTTGCGCAATTGGATTGGAAATGGGAACCCTAGTAAGCAACCAAAACTATGAGGCCATTGCTAATGCTATGCCTTCGCCGTTGGAAATAGAAACATATAGTGATCAGAGGGTTGAAAAATGGAGTGACAAAAGAATATGCCCACAATGGAGCCCTAATTCACAAGAGACCATTGTGCCTGAAAATCTCCCAAGATCATCTGCTCAAAGGAGATGGGAAACAGTTGGTTTTTCTAACGAGGATGCTCCGGCGGTTCAAATGGTAGTTAGAAAAGGTGGCAACTGCTTTGCTATGTAG SEQ ID NO: 20MTVTNFNCCLNPPPSNQSHGSSPSLPSKQNQVPAWNKNDHGSWAKRCVVGMSCIMIGFEMGSVVSNQTHEAIAKVMPLPLEIATSSDQRVAKWSEKRMCPQWSXNSLETIVPENLPRPSAQRRWEAVGFSKDAPAVQMVVRKGGNCFAM>ME00847G01190 (Manihot esculenta-1) SEQ ID NO: 21AAATTCAAAGAAAGGGTCAAGGCGAATGCGATTGCCTTGGCCGGGTTGAAGAACGACAAGTGGAGAAGCCAATGTTTACTGGGCATGGCATGCATCATAATTGGGCTTGAGATGGATTTGGCCAGCCATGAAAATCTTGCGGCGGCCGAAGATTTGCAATTTTCACTTGGGGAATCTAAGGAGAAAACCAAGAGATACAGATGGAGTGACAAAAGAATGTGTCCTCCATGGCGTCTTAATGCACTAGAGACCATTGTGCCTGAGAACCTACCAAGGCCATCAGCTCGACGGAGATGGGAGGCGATTGATTATTCAAAGATTGTTCCAGCTCCGGCTCCGGCAATTAAAGTGATAATCAGAAGCAGCAAGAATTGCTTTA CTATGTAASEQ ID NO: 22 KFKERVKANAIALAGLKNDKWRSQCLLGMACIIIGLEMDLASHENLAAAEDLQFSLGESKEKTKRYRWSDKRMCPPWRLNALETIVPENLPRPSARRRWEAIDYSKIVPAPAPAIKVIIRSSKNCFTM >ME04796G00360 (Manihot esculenta-2) SEQ ID NO: 23ATGGCCATTGCACCCAGTTGCTGCCTCAATCTCCGCCCTCCAACTCCACCCTCACCTCCTCCCAATGCAAGGGCTACCCAAGCTGCATGGTTCAAGAACGGCAGCTGGAGAAGCCAGTGTGTAGTGGGCATGGCCTGCATCATAATTGGAGTTGAAATGGATTTGGCGAGTCAAGCAAATGTTGCCACAGCCAAAGACTTGCAATATTTACTTGTAGAGTCGAAGGAGAACACCAAAGGTGACAGATGGAGTGACAGAAGAATTTGTCCTCCTTGGCATCTTAATTCGCTAGAGACCATTGTGCCGGAGAACCTTCCAAGGCCGTCGGCTCGTCGGAGATGGGAAGAGGTTGGTAATGTAAAGAATGTTCCGGCTCCGGCGATTAAAGTGATAGTTAAAAGCCGTAGCAGCAGCAACAATTGCTTTACCATGTAA SEQ ID NO: 24MAIAPSCCLNLRPPTPPSPPPNARATQAAWFKNGSWRSQCVVGMACIIIGVEMDLASQANVATAKDLQYLLVESKENTKGDRWSDRRICPPWHLNSLETIVPENLPRPSARRRWEEVGNVKNVPAPAIKVIVKSRSSSNNCFTM >MT3G107890 (Medicago truncatula)SEQ ID NO: 25 ATGACATCAACCAGTTGCTGCCTCCGTCTTTACCCTACAACTTCAAACGCTTCTCTCATCCCTAAAAACTCACCTCAACTTTCCTCGGAGATCAAAAACAGTGGATGCTGGAGAAGGCGGTGTGTTGTGATAGGAGTGGCTTCGTGCTTCTCTATAATTGGACTACAATTCAACAATTCAGTGTCATTGGAACATGAAGCTGTGGCTAAGGAGAATACCATGTTGGTGGCCATGTCAAATTCAATAGATGATGATGATGAGCATGTGTTTTTGGTTGGTGGTGCGGCCAAATGGAGCCAGAAAAGGATGTGCCCCTCTTGGCAAGGAAACAATCCCCTCGAAACCATCGTGCCAGAGAATCTTCCACGGCCAGCAGCACGTCGGAGATATGAGACTGTTCGCTCCACCTCTAAGATTGCTCCACCACTCTCAATGTCCGTCAAACTTAAAACCAATAGGGACAGTTGTTTCTCCATGTGA SEQ ID NO: 26MTSTSCCLRLYPTTSNASLIPKNSPQLSSEIKNSGCWRRRCVVIGVASCFSIIGLQFNNSVSLEHEAVAKENTMLVAMSNSIDDDDEHVFLVGGAAKWSQKRMCPSWQGNNPLETIVPENLPRPAARRRYETVRSTSKIAPPLSMSVKLKTNRDSCFSM >FQ394381 (Vitis vinifera)SEQ ID NO: 27 ATGGCCTTCTCTGCTGGTTGCTGCCTCAATCTCTCGCCTCCACCATCTGGGTCCAGCCCACGATCTTCTCGAAGCTCAACTAAAACTGATCAAGTTTCATGGCCAAGAAAAGAAAATTCATTGAAGAGCAAATGTCTCGTGGGGTTGACATGCATGATAATAAGCTTAGAAATGTCCAATTTAATGAGTGGTGAAGGGCTGGCCATTGCCCAAGATTTGCAATTAATTGGTGAAAGAAAAGAGGTAACGAGGTGGAGCGACAAGAGAATGTGCCCGCCCTGGCAGCTCAACTCATTGGAGACAATTGTGCCGGAGAACCTTCCCCGGCCGTCGACTCGCCGGAGATGGGAGTCAGTTGGTCATTCCACAACTGCCCCGGCAGTAAAAATTCTATTTAGAGCTCACACCAAGTCAGATTGTTTTTCCATGTGA SEQ ID NO: 28MAFSAGCCLNLSPPPSGSSPRSSRSSTKTDQVSWPRKENSLKSKCLVGLTCMIISLEMSNLMSGEGLAIAQDLQLIGERKEVTRWSDKRMCPPWQLNSLETIVPENLPRPSTRRRWESVGHSTTAPAVKILFRAHTKSDCFSM >OS05G49940 (Oryza sativa) SEQ ID NO: 29ATGGTGGTCTCCTGCCAGCTCAAGCCTGCGCCGGCTCCGGCCGCCGCCAGCAGAGGCGGCGGCGCGCCTCACCTCCAGCAGCTGCGCCGGGCGTGCGTCGCGGCGGCGGCGGCGTGCGCGGTGCTCGGGACGGCGGGCGGCCCCGGCGAAGGCGCCGTGATGGCGCGTGCGCCGGAGGCGACGGCGGCGGCGGCGGCGGGGCCGGCGCGGTGGAGCGACCGCCGGCAGTGCCCGCCGTGGCGCGCCAACTCGCTGGAGAACATCGTGCCGGAGAACCTGCCGCGGCCGTCGGCTCGCCGGAGGTTCAACAGCATCACGGCGGCGIGCGGCGGCGGAGAGCGCGCCGCCCCCCGCGTCGGCGTCGCCCGACGCCGTGCTCCCGTTCTTGGCGCCGCGCTCCGGCATGGGCTGCTTCTCCCTCTAA SEQ ID NO: 30MVVSCQLKPAPAPAAASRGGGAPHLQQLRRACVAAAAACAVLGTAGGPGEGAVMARAPEATAAAAAGPARWSDRRQCPPWRANSLENIVPENLPRPSARRRFNSITAAAAAESAPPPASASPDAVLPFLAPRSGMGCFSL >PT06G24730 (Populus trichocarpa)SEQ ID NO: 31 ATGGCCATCAGAACTACTTGTCGCCTCAATCTCTCCCCTCCAGGCTCTGGCTCAACCCTCCCTTCTTCCTCTACAAAGAACTCCCAGGTTGCCTGGTTCAAGAATGAAAAGTGGAGGAATCGATGTGTACTGGGCGCGGCGTGCATGATAATTGGACTTGAAATGGGAGGTGGTTTAGTGGGTGGTGAAGATCTTGCCATGGCTAGGGAGATGCAGGTGGCTGTGGAATCAAAAGAAAACTTGAATGGGCCAAGGTGGAGTGACAAGAGAATGTGCCCTCCATGGAGTCGGAATTCGCTAGAGACTATTGTGCCGGAGAACCTTCCAAGGCCATCGGCTCATAGGAGGTGGGAAGAAGTTCGCTTTTCCAAGAACAATGCTCCGGCCGTCAAAGTGATTGTGATCAAAAGAAGCAACGGTTGCTTCTCCATGTAA SEQ ID NO: 32MAIRTTCRLNLSPPGSGSTLPSSSTKNSQVAWFKNEKWRNRCVLGAACMIIGLEMGGGLVGGEDLAMAREMQVAVESKENLNGPRWSDKRMCPPWSRNSLETIVPENLPRPSAHRRWEEVRFSKNNAPAVKVIVIKRSNGCFSM >RC29912G02840 (Ricinus communis)SEQ ID NO: 33 ATGGCCATTACAACTAGTTGCTGCCTCAATATGAATATCCCTCCTCCAACTAGTGCTTCAAGTCTACCTTCTTCTTCTTCTACTACAAAGCCCACTGCTCAAGCCTCTTGGTTTCAAGAATGAGAAGTGGAGAAGCCAATGTGTACTAGGCATGGCCTGCATGATAATTGGACTTGAAATGGATAACTTGGTGAATGAAGAAACTAATCTTGCTATGGCCGCAGAGAATTCCTCATCGGTTGTAGAATTAAAGGTGAAACCAAAGACTAGAAGATGGAGTGATAAGAGAATGTGTCCTCCATGGAGGCTAAATTCACTAGAAACCATTGTGCCTGAGAATCTTCCAAGGCCATCAGCTCGTCGGAGATGGGAGGCTACTGGTTATTCTAAGATTGATCCGGCTCCGGCTCCGGCAAGGAAAGTGTCAGTCAAAAGCATTATGATTATGGATAATTGCTTTACCATGTAA SEQ ID NO: 34MAITTSCCLNMNIPPPTSASSLPSSSSTTKPTAQASWFKNEKWRSQCVLGMACMIIGLEMDNLVNEETNLAMAAENSSSVVELKVKPKTRRWSDKRMCPPWRLNSLETIVPENLPRPSARRRWEATGYSKIDPAPAPARKVSVKSIMIMDNCFTM>Solyc08g067630 (Solanum lycopersicum) SEQ ID NO: 35ATGGCTATTT CAACAAAGTT CTGCCTCAAT CTCTCCCCTC AACCTCCTCCTACTTCTAAT TATAATAACT CAATTCCCCC ACCTTCAAAA AAAACTCAACTTTCTTGGTA AGTCTACTAC TACTTTTTAC ATTTTTTTTT ATTTATCATACTTTGCTTTT CGTTTTTGGA TGTTTGTCTA TGTTAAAAGT CGTGTGCATCATGTCCATAT CTATTCATCC ATTTCAATTT ATGTGATATT ATATATGTTCATTCATCTGT TTCATTTTAT ACGACATTAT ATATATATAT ATATATATATATATACATTC AATTGTTTCA CTTTATATGA TTAAATTTTT TATTAATTTAAACTACTATT TTCATTTTTT TTCGCAGGCA ACGAAAAGAA AAATCATGGAAAAATCAATG TGTATTAGGA ATGGCATGTG TTGTAATTAT TGGATTAGAATTTGACGATT CAATTTTAGT TAATCAAGAA AGTACGATCG CGATCGCCGGAGACATGCAA TTACAATATG TCGCCGGAAA ATCAATACAA AAATGGAGTGAAAAAAGATC ATGCCCACCG TGGAACGTGA ACTCGTTAGA AACCATCGTGCCGGAAAACT TACCGAGGCC GGTGACTCGC CGGAGATGGG AAAACGTTGATTATAATACT ACTACTCAAT CTGCACCTGA AGTAAAGTTG GTGACAAAATTTAGTAAAGG ATGTTTCACT ATGTGA SEQ ID NO: 36MAISTKFCLNLSPQPPPTSNYNNSIPPPSKKTQLSWQRKEKSWKNQCVLGMACVVIIGLEFDDSILVNQESTIAIAGDMQLQYVAGKSIQKWSEKRSCPPWNVNSLETIVPENLPRPVTRRRWENVDYNTTTQSAPEVKLVTKFSKGCFTM >SB09G029300 (Sorghum bicolor-1)SEQ ID NO: 37 ATGGCGGTCTCCTCCATCAGCTGCTCCCTTCGGCCTCCAGCTCCCGTCAGAGAAGCTTCCGCTCGTCTGACGCCGCCGCAGCCGTCGCCACCAAAGACGACGGCCACGCCGTGGGCGGACGGGCTGCGGCGGGCATGCGTGGCGGCGGCGGCAACCGCGGCGTGCGTCGTGATCGGGACGGCGGGAGGTGGCGACGTGGTGGCGGCGTCGATGCCACGCGACACCCCCGTTCTGGCTGTGGACGCGCGGCCGGCGGCGGCGGCGCCGCGGTGGAGCGACCGCAGGGAGTGCCCGCCGTGGCGCGCTAACTCGCTGGAGAACATCGTGCCGGAGAACCTGCCCCGCCCGTCGGCGCGCCGGAGGTTCAACACAGTCAAGCGAGCGCCGCGGAAGGCCCCCGCGCTCGGGCGTCAAGCGGTGGCGCCGCCGTTCCTGGCGCTGCGCTCCGGCGTGGACGACTGCTTCACCCTCTAG SEQ ID NO: 38MAVSSISCSLRPPAPVREASARLTPPQPSPPKTTATPWADGLRRACVAAAATAACVVIGTAGGGDVVAASMPRDTPVLAVDARPAAAAPRWSDRRECPPWRANSLENIVPENLPRPSARRRFNTVKRAPRKAPALGRQAVAPPFLALRSGVDDCFTL>SB09G029310 (Sorghum bicolor-2) SEQ ID NO: 39ATGGCGGCTTCCTCCACCGCCACCACCATAATCAGCTGCTGCTGCTGCTGCCTCGGGCCTCCCGCTCCGCCCAAAGAATCCTCTGCAGGCGCTCGCAGGCCGCAGGCGCCGGCAGGCGTGTCAGTGTCGTCGCACGCGCTGCGCCGGGCGTGCGTGGCTGCCGCGGCGTGCGCGATGGTGGGGATTTCGGGCGGCGGCGGCGGCGCCGACATGGCCCTTGCGCTGGCGCGTGGCGGCGGCGCGTTCGCCTCCAGGACCGACGTCGTCGCCGTGTCCGTGGGCGCCGCGCGCGCCAAGGCGCCGCCGCGGTGGAGCGACCGCAGGCAGTGCCCGCCGTGGCGCGCCAACTCGCTGGAGAACATCGTGCCGGAGAACCTGCCCCGCCCGTCCGCGCCCAGGAGGTTCGACAGCGTCTCGGCCTCGGCGGCCGCGCCGGACTTGTCGGCGCCGCCTTCTTTCCTGGCGCTGCGACCCGGCACGGGCTGCTTC TCACTCTGASEQ ID NO: 40 MAASSTATTIISCCCCCLGPPAPPKESSAGARRPQAPAGVSVSSHALRRACVAAAACAMVGISGGGGGADMALALARGGGAFASRTDVVAVSVGAARAKAPPRWSDRRQCPPWRANSLENIVPENLPRPSAPRRFDSVSASAAAPDLSAPPSFLALRPGTGCFSL>TC09G013320 (Theobroma cacao) SEQ ID NO: 41ATGGCCATTTCAACTAGGTGCTGCCTCAATGTGTCCCCTCCAACTCCAACTCCTGGCTTTGACATGTCTTCTTCTAACAAGAAGGCATCCCAAGTTGCATGGCCAAGGGATGATAAATGGAGGAAGCAATGTGTACTAGGGGTAACCTGCATCGTAATTGGATTACAAGTAGGTAATATAACTGACAACAGCGCCATTGCTGAGGAAGTCTCATCTGCCACAGAGTCAAACTCGAAAGTAGCAAGATGGAGTGATAAAAGAGTGTGCCCTCCATGGAATGCAAATTCGCTGGAGACCATCGTGCCGGAGAATCTCCCACGACCATCAGCTCATAGAAGATGGGAAGCTATTGGTTTCTCCAAGAATGCCCCGGCAGTCAGAGTGAAAGTGACAACAAAAACAAGAACCAATTGCTTCTCCATGTAA SEQ ID NO: 42MAISTRCCLNVSPPTPTPGFDMSSSNKKASQVAWPRDDKWRKQCVLGVTCIVIGLQVGNITDNSAIAEEVSSATESNSKVARWSDKRVCPPWNANSLETIVPENLPRPSAHRRWEAIGFSKNAPAVRVKVTTKTRTNCFSM >AZ916442 (Zea mays) SEQ ID NO: 43CCGTTTCACCGTTATATCTGCAGGGCGGACGGGCTGCGGCGGGCGTGCGTGGCGGGCGCGGCGGCGTGCGTCGTGTTCGGGACGGCGGGAGGCGGCGGCGGCGGCGTGGCCGCGTCGGCGCCGCCGCGCGACGCCTCCGTCGCGGCGGCCCCGCGGTGGAGCGACCGCCGGGAGTGCCCGCCGTGGCGCGCCAACTCGCTGGAGAACGTCGTGCCGGAGAACCTGCCCCGCCCGTCGGCGCGCCGGAGGTTCAGCACCGTCAAGCGGGCGCCGCGGAAGGCCCCCGCGCTCGGGCCTCAGGCGGTGGCGCCGTCGCCGTTCCTGGCGCTGCGATCCGGCATGGACGACTGCTTCACCCTC SEQ ID NO: 44PFHRYICRADGLRRACVAGAAACVVFGTAGGGGGGVAASAPPRDASVAAAPRWSDRRECPPWRANSLENVVPENLPRPSARRRFSTVKRAPRKAPALGPQAVAPSPFLALRS GMDDCFTLSEQ ID NO: 45 RxCxxWxxN SEQ ID NO: 46 ExxxPENLPRxxxxxR SEQ ID NO: 47TTACACGTAATGAACTTCCAG

1. A method of preparing a transgenic plant having enhanced stresstolerance, the method comprising (a) introducing into a population ofplants an expression cassette that inhibits expression of a chloroplastvesiculation (CV) gene; and (b) selecting a plant having enhanced stresstolerance compared to a control plant that does not comprise theexpression cassette.
 2. The method of claim 1, wherein the expressioncassette comprises a nucleic acid sequence encoding a microRNA or ansiRNA specific to the CV gene.
 3. The method of claim 1, wherein the CVgene encodes a CV protein comprising a consensus sequence as shown inSEQ ID NO: 45 or SEQ ID NO:
 46. 4. The method of claim 1, wherein the CVgene encodes a polypeptide comprising an amino acid sequence at least90% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or
 44. 5. The method ofclaim 1, wherein the step of introducing is carried out usingAgrobacterium.
 6. The method of claim 1, wherein the expression cassettecomprises a constitutive promoter.
 7. The method of claim 1, wherein theexpression cassette comprises an inducible promoter.
 8. The method ofclaim 7, wherein the inducible promoter is induced in response to stressconditions.
 9. The method of claim 1, wherein the transgenic plant hasenhanced tolerance to an abiotic stress.
 10. The method of claim 9,wherein the abiotic stress is high salt conditions and the step ofselecting includes selecting plants having enhanced salt tolerance. 11.The method of claim 9, wherein the abiotic stress is drought conditionsand the step of selecting includes selecting plants having enhanceddrought tolerance.
 12. A plant prepared by the method of claim
 1. 13. Anisolated nucleic acid molecule comprising a plant promoter operablylinked to a heterologous nucleic acid sequence encoding a microRNA or ansiRNA specific to a target CV gene.
 14. The isolated nucleic acidmolecule of claim 13, wherein the target CV gene encodes a CV proteincomprising (i) a consensus sequence as shown in SEQ ID NO: 45 or SEQ IDNO: 46 or (ii) an amino acid sequence at least 90% identical to any oneof SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, or
 44. 15. The isolated nucleic acid molecule ofclaim 13, wherein the plant promoter is a constitutive promoter.
 16. Theisolated nucleic acid molecule of claim 13, wherein the plant promoteris an inducible promoter.
 17. A method of preparing a transgenic planthaving enhanced stress tolerance, the method comprising (a) introducingmutations in CV genes in a population of plants; and (b) selecting aplant having enhanced stress tolerance compared to a control plant thatdoes not comprise the mutation.
 18. The method of claim 17, wherein themutations are introduced using chemical mutagenesis.
 19. The method ofclaim 18, wherein mutations in CV genes in the population of plants areidentified using Targeting Induced Local Lesions in Genomes (TILLING).20. A plant prepared by the method of claim
 17. 21. A transgenic plantcomprising an expression cassette comprising a plant promoter operablylinked to a nucleic acid sequence encoding a microRNA or an siRNAspecific to a target CV gene.
 22. The transgenic plant of claim 21,wherein the target CV gene encodes a CV protein comprising (i) aconsensus sequence as shown in SEQ ID NO: 45 or SEQ ID NO: 46 or (ii) anamino acid sequence at least 90% identical to any one of SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, or
 44. 23. A method of preparing a transgenic plant having enhancednutrient assimilation, the method comprising (a) introducing into apopulation of plants an expression cassette comprising an inducibleplant promoter operably linked to a heterologous CV polynucleotidesequence encoding a CV protein comprising (i) a consensus sequence asshown in SEQ ID NO: 45 or SEQ ID NO: 46 or (ii) an amino acid sequenceat least 90% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44; and (b)selecting a plant having enhanced nutrient assimilation compared to acontrol plant that does not comprise the expression cassette.
 24. Themethod of claim 23, wherein the step of introducing is carried usingAgrobacterium.
 25. The method of claim 23, wherein the step of selectingis carried out by selecting plants with increased fruit yield comparedto control plants.
 26. A transgenic plant prepared by the method ofclaim
 23. 27. A transgenic plant comprising an expression cassettecomprising an inducible plant promoter operably linked to a heterologousCV polynucleotide sequence encoding a CV protein comprising (i) aconsensus sequence as shown in SEQ ID NO: 45 or SEQ ID NO: 46 or (ii) anamino acid sequence at least 90% identical to any one of SEQ ID NOs: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, or
 44. 28. An isolated nucleic acid molecule comprising anexpression cassette comprising an inducible plant promoter operablylinked to a CV polynucleotide sequence encoding a CV protein comprising(i) a consensus sequence as shown in SEQ ID NO: 45 or SEQ ID NO: 46 or(ii) an amino acid sequence at least 90% identical to any one of SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, or 44.